Strategic Breeding Approaches: Harnessing Molecular and Phenotypic Markers to Overcome Quinoa's Flower Morphology Bottlenecks

Strategic Breeding Approaches: Harnessing Molecular and Phenotypic Markers to Overcome Quinoa's Flower Morphology Bottlenecks | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Strategic Breeding Approaches: Harnessing Molecular and Phenotypic Markers to Overcome Quinoa's Flower Morphology Bottlenecks Prashant Vikram, HIFZUR RAHMAN, Lovely Mehta, Sakshi Vidit Jain, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9391447/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Quinoa represents a valuable gift from the "New World" to the "Old World." Although some breeding programs exist worldwide, they have seen limited growth and impact on its genetic improvement. Large-scale hybridization, hindered by the crop's complex floral morphology, remains the primary bottleneck in quinoa breeding. This study compared manual and natural crossing schemes, incorporating hybridity testing with morphological and molecular markers. The results indicate that a facilitated open-pollinated strategy combined with hand emasculation and hybridity confirmation using morphological or indel markers offers a feasible approach to establishing a quinoa breeding pipeline. We also concluded that characterising panicles by the arrangement of hermaphrodite and pistillate flowers could be a game-changer for improving breeding efficiency. Additionally, interspecific crosses (Chenopodium quinoa × Chenopodium giganteum) achieved efficiencies of 5–26%. Both inter- and intraspecific crosses can feasibly generate variation for genetic improvement in quinoa. Breeding quinoa for Asian environments should follow a step-wise strategy: (1) characterize flowering in crossing blocks, (2) apply natural crossing or hand emasculation, (3) confirm hybridity using morphological or molecular markers, (4) pursue genetic enhancement, and (5) select for traits of interest. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Genetics Biological sciences/Plant sciences Quinoa Pseudocereals breeding marker-assisted breeding genetic enhancement Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Quinoa ( Chenopodium quinoa ) is one of the oldest crops of the American continent, believed to have originated and cultivated in the Andean region, mainly in Peru, Bolivia, and Chile. Quinoa can be considered a new gift of the ‘new world’ to the ‘old world’, like the potato and tomato. Quinoa has earned special attention worldwide due to its exceptional nutritional quality, health benefits and ability to adapt to marginal environments (Jacobsen, 1997, 2003, 2015). Quinoa, a pseudocereal due to its grain structure, is an annual dicotyledonous species that belongs to the family Amaranthaceae (formerly Chenopodiaceae ). Quinoa is exemplary for amino acid composition and offers a remarkable balance of oil (4–9%), protein (averaging 16%, with high nutritional value), and carbohydrates (64%) (James 2009). It can be used to make flour in the same way as cereals due to its high starch content (51–61%). Quinoa grain is free of gluten, which has facilitated the creation of various products for people with celiac disease (i.e., those who are allergic to gluten) (Singh 2019, Amin et al. 2022). Quinoa is one of the oldest crops but its limited spread happened in the past few decades (Bazile et al. 2016). The major reason for the limited spread is primitive breeding efforts, mostly reported from Bolivia and Peru (Zurita-Silva et al. 2014). Although some recent breeding studies have reported the development of high-yielding varieties adapted to temperate regions and high latitudes of Europe, North America, and China, most of these were limited to mutation breeding or mass selection (Murphy et al. 2016, Patirange et al. 2022). Many Asian and African regions are continuously making efforts to increase the genetic biodiversity of the quinoa cropping system by hybridization to develop high-yielding, sweet and improved varieties, with limited success (Murphy et al. 2016). The biggest challenge in large-scale quinoa breeding is due to its very small florets and the presence of both hermaphrodite and female flowers (out-crossing 0.5% to 17%) on the same panicle (Murphy et al. 2016). Due to the complex arrangement of the hermaphrodite and pistillate (female) flowers, small size of flowers and fast progression of flowering within infloresence it becomes very difficult to perform hand emasculation efficiently (Paterson et al. 2015). Hot water treatment is another method followed for emasculation in which hot water (~ 45°C) application is done on the inflorescence in a way to inactivate the pollens while keeping ovaries and stigma safe, however, this method is also reported to damage the inflorescence (Fleming et al. 1995, Sha 2013). Though it is time-consuming, imparting male sterility can be an alternative method of creating hybrids. Some male sterility sources have been identified in quinoa including cytoplasmic male sterility (CMS) germplasms along with their restorer genes (Simmonds 1971, Ward and Johnson 1994). It is important to note that heterosis has not been reported in quinoa so far, so, application of CMS system might not be very feasible option, hence not pursued further at commercial scale. Quinoa is also known for its highly variable outcrossing rates across different accessions (Murphy et al. 2016) that can be utilized for enhancing the quinoa breeding program efficiency. Identification of the true F 1 s is another important concern in quinoa breeding. Broadly, there are two strategies implied for the identification of F 1 plants in quinoa i.e. using morphological and/or molecular markers. Traits such as axillary pigmentation, plant colour, seed colour and inflorescence can be used as diagnostic markers provided the male parent (pollinator) is homozygous for the dominant alleles (Paterson et al. 2015). On the other hand, molecular marker use in hybridity testing requires polymorphic markers between the two parents of the F 1 plants. In the current study, efforts have been made to employ different crossing strategies to establish a robust quinoa breeding platform specifically for the newer environments including Asia/ Middle East. 2. Material and Methods 2.1. The quinoa crossing block Quinoa breeding work was initiated using gene bank accessions stored in the International Centre for Biosaline Agriculture (ICBA), Dubai, UAE, in December 2020. A subset from these accessions was subjected to saponin estimation analysis and reported recently (Tabatabei et al. 2022). Low saponin accessions were shortlisted from the report of Tabatabaei et al. 2021, and, validated through reanalysis following afrosimetric method (Koziol 1991). Seven parents were found to be suitable donors for low saponin parents (Ames 19046, D12401, D12406, D 11912, D12377, Chen281 and Chen254). Based on visual observations, six other donors (Co 407, D12047, ICBA-Q5, BO 51, Titicaca and ICBA-Q3) were selected for grain yield and adaptive characteristics (Supplementary Figs. 1a-b). ICBA-Q5 and ICBA-Q3 are two lines selected at ICBA and were nominated as varieties in different countries ( https://www.biosaline.org/news/2020-03-14-7046 ). Titicaca is a widespread variety of Europe; Co 407 is a variety from Colorado State University, USA, developed by mutation. D12047 and BO 51 were identified as high-yielding accessions during field experiments for the GWAS study at ICBA fields in the 2020-21 trial (Rahman et al. 2024). 2.2. DNA extraction and PCR amplification Genetic analyses were performed for the characterization of crossing blocks and testing of hybridity. DNA of the crossing block lines and F 1 plants were extracted following a modified CTAB method explained by (Cota-Sanchez et al. 2006). The InDel markers reported by (Zhang et al. 2017) were synthesized, and a parental polymorphism survey was carried out on the parental DNA. DNA was amplified using protocol explained by (Collar and Mackill, 2009). Reaction mixture of 20 µL included 10 µL of master (2X FIREPol® master mix), 0.5 µL of (0.1 µM) for each primer, and 1 µL of genomic DNA (50 ng/µL). The final volume was obtained using sterile distilled water. The PCR reaction was performed using a thermal profile with pre-denaturing for 5 min at 95°C, followed by 40 cycles at 94°C for 40 s, annealing for 40 s, and extension at 72°C for 30 sec, with a final extension time of 5 min at 72°C. PCR products were separated on a 3.0% agarose gel. 2.3. Genetic analysis Data were scored as (1) for presence and (0) for absence for each sample under investigation. The polymorphism information content (PIC) was calculated according to the formula: PIC = 1 − Σ pi 2 where pi is the frequency of the i th allele of the locus in eight genotypes (Anderson et al. 1993).The data was analysed with SIMQUAL program of NTSYS-pc (Version 2.02), and similarities between accessions were estimated using the Jaccard’s coefficient calculated as J = A / (N - D), where A is the number of positive matches (that is, presence of band in both samples), D is the number of negative matches (that is, absence of band in both samples) and N is the total sample size including both the number of matches and unmatched. A dendrogram was created from the resultant similarity matrices using the UPGMA (Unweighted Pair Group Method with Arithmetic mean) method following the SAHN function of NTSYS-pc (Version 2.02). 2.4. Crossing and population advancement Making crosses in quinoa is challenging because of its gyno-monoecious flowers and high resemblance of the hermaphrodite and female ones. Three different approaches were followed for making crosses by: (1) removing hermaphrodite flowers from the female parents, (2) forced synchronization of flowering and (3) natural crossing. 2.4.1. Crossing by removing hermaphrodite flowers All 13 parents were sown in staggered planting mode in ICBA’s green house during 5th December 2020 to 31st January 2021 at 15 days interval. At the peak flowering stage, hermaphrodite flowers were removed from the inflorescence leaving only female flowers on female parents and clubbed with that of male parent (in which hermaphrodite flowers were not removed) and covered with a glassine bag as illustrated in Supplementary Figs. 2 (a) and (b). Hermaphrodite flowers were observed and identified using magnifying glasses (Supplementary Fig. 2c). One of the 13 parents was Chenopodium giganteum (Ames 19046) which was crossed with four Chenopodium quinoa accessions viz. D-12406, ICBA-Q3, Chen-254 and D-12377 (Supplementary Fig. 2b). 2.4.2. Forced synchronization of flowering Five genotypes were identified for crossing through controlled flowering to force synchronization of anthesis: 1. ICBA-Q5, 2. D12401, 3. D12406, 4. Co 407 and 5. D12377. Selection of these five genotypes was done based on their unique characteristics including D12401: purple colour hermaphrodite flower, D12406: similar to D12401 but white colour hermaphrodite flowers, D12377: black seeded, Co 407: purple panicle and ICBA-Q5: Very early maturing. These five genotypes were sown in a clockwise fashion in the pot starting from #1 to #5 (Fig. 1 ). They were first allowed to grow vegetatively, however, during flowering stage, early flowering panicles were cut with scissors in order to synchronize their flowering with other lines (Supplementary Fig. 2d). Quinoa plant has characteristic of continuously flowering for several weeks. This characteristic was exploited in this approach. We continued synchronization until when majority of the five plants flowered synchronously. After manual removal of panicles from the top, plants became usually short heighted and tender so that it became easy to tie (loosely) all of them and cover with a big glassine bag. Later on, each of the five lines were harvested from different pots and bulked. Seeds of individual five lines were sown in the next season in single plots so that crosses individuals can be visualized with the help of their parental distinctive diagnostic characteristics. 2.4.3. Natural crossing strategy through no emasculation method In another approach, the pots with five genotypes (D12401, D12406, D12377, Co 407 & ICBA-Q5) with distinctive morphological characteristics as explained in the above section were kept just in front of the cooling pad of greenhouse so that slowly blowing wind from fan can facilitate movement of pollen from one genotype to another (Supplementary Fig. 2e). This way natural out crossing was facilitated. Each of the five genotypes were harvested from all pots and bulked for the next season sowing. As mentioned above, each line was harvested individually from different pots and formed five different bulks. Seeds of individual bulks were grown in the next season in individual plots to visualize probable crosses with the help of distinctive diagnostic characteristics. This method presents a slight modification in the ´no emasculation method´ as reported by (Emrani et al. 2020). 3. Results 3.1. Saponin estimation of the crossing block Based on the saponin estimation analysis according to using afrosimetric method (Michael J Koziol 1991) parents for quinoa crossing block were declared. A total of seven genotypes were found suitable to use as saponin free donor parents (Ames 19046, D12401, D12406, D 11912, D12377, Chen281 and Chen254), whereas six genotypes were used as parents for high grain yield and adaptive characteristics (Co 407, D12047, ICBA-Q5, BO 51, Titicaca and ICBA-Q3) based on in house experiments (ICBA Unpublished). Results are depicted in Supplementary Figs. 1(a) and (b). Unique characteristics of all the parents are delineated in the Table 1 . Table 1 Salient characteristics of the parents used for quinoa crossing block Genotype Name Key Trait Remarks Ames-19046 Saponin free-Super late Preferred for Forage, different grain and plant type. Chen-254 Saponin free Medium Chen-281 Saponin free Medium D-11912 Saponin free Medium D-12377 Saponin free This is a heterogenous line producing two types of seed (brown & black), its f loral biology and flowering pattern need to be studied. D-12401 Saponin free Medium, this line has diagnostic hermaphrodite flower in certain environmental condition (pink color hermaphrodite flower, if grown in open light condition). D-12406 Saponin free Medium BO-51 Yield & Adaptation Traits Early Co-407 Yield & Adaptation Traits Early D-12047 Yield & Adaptation Traits This might be a segregating line, need not to use in breeding further. ICBA-Q3 Yield & Adaptation Traits Late ICBA-Q5 Yield & Adaptation Traits Early Titicaca Yield & Adaptation Traits Very Early 3.2. Genetic profiling of parents in crossing block Out of 36 indel primers studied 25 indels showed polymorphism among different accessions of crossing block. Figure 2 presents relationship among different crossing block accessions. Dendrogram clearly reveals that Chenopodium giganteum (Ames 19046) is distantly related with Chenopodium quinoa genotypes of crossing block. Broadly, there were two groups i.e. GP-I and GP-II; GP-I had three saponin free lines Chen254, D12406 and D 11912, whereas GP-II included ten diverse accessions including Ames 19046, D12401, D12377, Chen281, Co 407, D12047, ICBA-Q5, BO 51, Titicaca and ICBA-Q3. 3.3. Efficiency of three crossing methods The hybridity testing of 15 cross combinations ascertained through seeds obtained from female flowers after removing hermaphrodite flowers from female parents, crossing efficiency was found in range of 5 to 71% (Table 2 , Figs. 3 a-c). In the interspecific crosses of ‘ Chenopodium quinoa × Chenopodium giganteum ’ this Figure ranged 5–26%, whereas, among ‘ Chenopodium quinoa × Chenopodium quinoa ’ crosses it was 10–71%. While making interspecific crosses Chenopodium giganteum was used as male parent because of its floral arrangement and multiple hanging branches. Among four interspecific crosses, ‘ICBA-Q3 × Ames-19046’ did not produced viable seed, whereas, other three successfully produced viable seeds. Maximum number of true hybrids were observed in ‘D-12401 × BO-51’ cross i.e. 15 out of 21 (71%), and minimum crossing efficiency was revealed in ‘D-12401 × ICBA-Q3’ as presented in Table 2 . Table 2 Table presenting the bi-parental crosses confirmed with Indel-based markers, crossing efficiency has been presented with respect to individuals tested in the laboratory. Cross Type Ref# Cross Name No. of true hybrids Total number of seed Crossing Efficiency (%) Crossing Date Chenopodium quinoa × Chenopodium giganteum A14 D-12406 × Ames-19046 1 20 5 03/02/2021 C-18* ICBA-Q3 × Ames-19046 1 15 7 03/01/2021 A3 Chen-254 × Ames-19046 2 13 15 03/08/2021 A2 D-12377 × Ames-19046 6 23 26 03/08/2021 Chenopodium quinoa × Chenopodium quinoa B-11 D-12401 × ICBA-Q3 1 10 10 14/02/2021 AC-5 ICBA-Q5 × D-12377 2 11 18 04/07/2021 C-4 ICBA-Q5 × ICBA-Q3 1 4 25 24/02/2021 AC-8 BO-51 × D-12377 3 12 25 04/11/2021 B-37 D-12377 × ICBA-Q5 2 5 40 03/14/2021 B-42 D-12406 × ICBA-Q5 2 5 40 03/15/2021 A8 D-12377 × Chen-281 11 24 46 04/07/2021 C-9 Titicaca × D-12377 1 2 50 02/08/2021 B-44 D-12406 × ICBA-Q3 9 17 53 03/01/2021 B-25 D-12401 × ICBA-Q5 8 12 67 04/14/2021 B-24 D-12401 × BO-51 15 21 71 04/14/2021 Among five parents used for natural crossing, only D12377 was black seeded parent, and this trait was used for determining the efficiency of natural crossing. There were total of 10 pots each having five parents for natural crossing purpose including D12377 as one of them. All the D12377 plants (i.e. 10 plants, one from each pot) were harvested and seed was bulked. A total of 1000 seed were sown to get 823 plants from naturally crossed black seeds (produced with D12377 as female parent). Among 823 plants, 205 and 618 produced white and black seeds respectively. As mentioned above, the 1000 seeds sown from 10 different supposedly, naturally crossed D12377 plants (i.e. we are not sure which of them underwent natural crossing). In way to analyse the crossing efficiency, we here assume that the 618 plants producing black seeds came from selfed black seeded D12377 and vice-versa for the rest 205 white seed producing plants. Therefore, with this assumption (618 black seeded plants are from selfed D12377 parent), we can inference that 205/618 × 100 ~ 25% would be the minimum crossing efficiency. It is important to note here that this efficiency was achieved under a specific situation i.e. with five plants sown in a pot and kept together in very closed vicinity and 618 black seeded plants were assumed to be derived from selfed plants (Fig. 4 ). Manual observation based on distinctive diagnostic characteristics of the five parents subjected to forced flowering synchronization, crossing efficiency was observed in range from 1.9% in D12406 (5/263) to 7.7% in ICBA-Q5 (6/78). In D12401 (16/410) and D12377 (6/154) efficiency was approximately 4% (data not presented). 3.4. Inter-specific cross compatibility Among four inter-specific crosses attempted in our study with Chenopodium giganteum (Ames 19046) as common male parent and Chen254, D12406, D12377 and ICBA-Q3 as females (Supplementary Figs. 3a-b). F 1 seeds of one cross (i.e. Ames 19046 × ICBA-Q3) did not produced F 2 seeds (Supplementary Fig. 3c). The other three crosses, Ames 19046 × Chen254, Ames 19046 × D12406 and Ames 19046 × D12377 successfully produced F 2 seeds. 3.5. Morphological marker identification With respect to arrangement of hermaphrodite and female flowers, two variants were observed that could serve as morphological markers. The low saponin parent D12401 was identified most suitable for crossing because of its pink coloured hermaphrodite flowers which were clearly distinct from the female ones (Fig. 5 ). Similarly, in one of the high saponin line D12047, hermaphrodite and female flowers were arranged in somewhat conical fashion, in which hermaphrodite flower was placed on the tip and female ones on side (Supplementary Fig. 4). F 1 derived from cross of D12377 × ICBA-Q5 was identified as a distinctive diagnostic panicle colour as illustrated in (Supplementary Fig. 5). In the late maturity stage, panicle colour of D12377, ICBA-Q5 and F 1 were clearly distinct. Similarly, in the natural crossing scheme, five characteristically distinct genotypes (Co 407, D12401, D12406, D12377 and ICBA-Q5), were harvested and individually bulked. Characteristics of D12377 are green panicle and black seed colour. Another parent Co 407 has purple coloured panicle which produced white seeds. Therefore, if a F 1 plant derived from the cross of these two accessions might inherit at least one trait from each parent - D12377 and Co 407. Figure 6 presents a panicle of F1 plant in which the panicle colour is purple (similar to C0 407) producing black seed (trait from D12377). This indicates that the F 1 was produced from pollination of female flower of Co 407 by hermaphrodite flowers of D12377 or vice-versa. 3.6. Seed colour observations in quinoa F 1 seeds derived from cross of five white seed (female) with one black seed parent (male), were all white i.e. female parent type. The F 2 seeds produced on all these five F 1 s were found black coloured. Figure 7 presents the seed colour of parents (Titicaca, ICBA-Q5, ICBA-Q3, BO 51 and D12377) and F 2 seeds. Seeds of fifth cross of Chen281 (white seeded) × D12377 showed similar pattern (less seeds were produced and advanced immediately, picture couldn’t be taken). These results indicated toward dominance of the black seed colour over white in quinoa. However, to better understand this pattern reciprocal crosses were made using white seeded female (ICBA-Q5) with black seeded male (D12377) and vice-versa. In both cases F 1 seeds tend to be maternal type which clearly revealed maternal inheritance, not the dominance of any seed coat colour (Fig. 8 ). Therefore, existence of white coloured F 2 seeds on F 1 plants made from the cross of white seeded male (ICBA-Q5) with black seeded female (D12377) rules out any conclusion on dominance of seed colour in quinoa. Further, 76 F 2 seeds of each of the two populations Titicaca × D12377 and Chen281 × D12377 were advanced and colour of F 3 seeds were observed to better understand segregation pattern. The 76 F 2 plants of Titicaca × D12377 produced 53 black and 23 white seeds in the ratio of 2.3:1. Similarly, 76 F 2 plants of Chen281 × D12377 produced 51 black and 25 white seeds in the ratio of 2.07:1. Chi squared test performed in both crosses ruled out the significance of Mendelian monohybrid ratio of 5:3 in F3 generation indicating lack of complete dominance of black seed colour in quinoa (Table 3 ). Table 3 Table presenting the chi-squared test to test “the goodness of fit” with the null hypothesis (H 0 ): Ratio of Black and White seeds in F3 generation were 5:3. The F 2:3 seeds were produced by two populations i.e. ‘Titicaca × D-12377’ and ‘Chen 281 × D-12377’ Titicaca × D-12377 Black White Total 53 23 76 Chen 281 × D-12377 51 25 76 Chi-Square calculation S.N. Observed (O) Expected (E) O-E (O-E) 2 (O-E) 2 /E 1 53 47.5 5.5 30.25 0.63684 2 23 28.5 -5.5 30.25 1.0614 3 51 47.5 3.5 12.25 0.25789 4 25 28.5 -3.5 12.25 0.42982 Chi-Squared value 2.38 The chi-Squared value at α = 0.05 with degree of freedom = 1, the chi-squared value was 3.841. The calculated chi-Squared value was 2.38 which was lesser than 3.841 with 5% significance level. Therefore, we can reject the null hypothesis. 4. Discussion Importance of quinoa outside its domestication centre i.e. Andes with ensured irrigation has been recognised in past few years. Quinoa can also be introduced/ scaled up in different parts of Asia and Africa by transforming the existing cropping systems and developing suitable varieties that can perform well under specific environments. Quinoa breeding work to develop the improved varieties is being undertaken at ICBA, UAE successfully. 4.1. Inter-specific crosses in Chenopodium species : Opportunity of exploiting variation Defining a crossing block of diverse genotypes is the first step in establishing a breeding program. Low saponin, early maturity and high yield were selected as the three most important traits required for the adaptation of quinoa in newer environments. Thirteen parents in crossing blocks comprised of six Chenopodum quinoa and one Chenopodum giganteum accessions with saponin free/ very low saponin content along with six other high yielding varieties/ genotypes (Supplementary Figs. 1a-b). Diversity pattern of these 13 genotypes (Fig. 2 ) based on similarity index could be the best way to choose potential parents. Interestingly, F 1 plants from three interspecific crosses made with ‘saponin free Chenopodum giganteum ’ with ‘saponin free Chenopodum quinoa ’ successfully produced F 2 seeds, whereas F 1 of ‘saponin free Chenopodum giganteum ’ × ‘high saponin Chenopodum quinoa ’ did not produced F 2 seeds. Noticeably, the plant type of F 1 s developed from inter-specific crosses ( Chenopodum quinoa × Chenopodum giganteum ) resembled respective Chenopodum quinoa parent (D12406, D12377 and Chen254), and successfully produced the F 2 seeds. Conversely, plant type of F 1 developed from ICBA-Q3 ( Chenopodum quinoa ) × Ames 19046 ( Chenopodum giganteum ) resembled Chenopodum giganteum parent (Ames 19046) and did not produce the F 2 seeds (Supplementary Fig. 3c). These observations clearly suggest that inter-specific crosses with Chenopodum quinoa with other Chenopodum spp. can be exploited for the desired genetic advance. The inter-specific cross compatibility and heterosis analyses can be performed at a large scale for identification of suitable genetic variants. In order to ensure adaptability of Chenopodum quinoa in newer environments, genetic variations created from inter-specific crosses can play a major role. 4.2. Role of distinctive phenotypic traits as diagnostic markers in natural crossing Among 13 crossing block accessions, two distinctive diagnostic floral arrangements were observed that can greatly help in the manual crossing process. The pink-coloured hermaphrodite flowers with green-coloured female ones in quinoa genotype D12401 (Fig. 5 ) and the positioning of hermaphrodite flowers on the top of a cone, while female ones on sides in D12047 (Supplementary Fig. 4) render them very easily discriminated from female flowers. These flowers can, therefore, be easily removed or destroyed and female ones can be pollinated with other accessions. The black seed colour was observed as another diagnostic characteristic that can be potentially used in natural crossing. Five quinoa genotypes underwent natural crossing (or out-crossing), as presented in Fig. 1 and were harvested individually and grown in separate plots in the next generation i.e. one genotype in one plot. True hybrids were identified successfully with the help of seed colour of F 1 seeds. If black-seeded plants produced white seeds or vice-versa were declared as true hybrids. Similarly, the purple panicle colour of Co 407 was used as a diagnostic characteristic while crossing quinoa genotypes naturally. However, in every breeding program, hybridity testing using polymorphic markers is always advisable to employ as fail-safe technology. The probability of identifying the desired genetic variant increases by enhancing the number of crosses and population sizes. Therefore, a feasible crossing methodology ensuring a large number of crosses with minimal effort should help quinoa breeding greatly. The efficiency of three different crossing methods indicated the importance of quinoa germplasms with distinctive diagnostic characteristics, such as black seed colour and purple panicle. It was very interesting to note here that the outcrossing efficiency of the black-seeded quinoa line, D12377 was observed to be 25%, assuming that all other black seeds were selfed. Out of 823 putative F 1 plants taking D12377 as female parent (black seeded), 205 produced white seeds and rest 618 yielded black seeds (Fig. 4 ). We did not observe any plant producing both black and white seed. The forced flowering synchronization method attempted in this study did not provide encouraging results, possibly because of trimming large number of flowers for synchronization. Manual crossing through removing hermaphrodite flowers, revealed a wide range of success rate indicating the role of varying floral arrangements among quinoa germplasm accessions. In nutshell, a large-scale effort in identification of distinctive diagnostic traits and floral biology in quinoa can help greatly in creating desired genetic variants that may further help in its wider adaptation in newer environments of Africa and Asia. 4.3. Marker assisted breeding: Tool that can handle crossing efficiency issue in quinoa It is a well-known fact that due to gyno monoecious nature of flowers in quinoa, manual emasculations become difficult and less efficient. Therefore, efforts have been made in past to emasculate using different approaches including manual emasculation and hot water treatments (Emrani et al. 2020, Peterson et al. 2015, Sagar et al. 2025). However, establishing breeding pipeline and making large-scale efforts would require feasibly applicable alternatives. In this study we have presented that hybridity testing of the putative crosses through mechanical emasculation (like removal of hermaphrodite flowers) as fool proof backup strategy using abundantly available indel polymorphic markers in quinoa (Fig. 3 , Table 2 ). Therefore, the bottleneck of low efficiency of manual crossing in quinoa can be effectively addressed by using marker assisted breeding approaches. The aforesaid methods of crossing could be used to fast-track breeding efforts in difficult crop like quinoa, in conjunction with hybridity testing using indel markers as fool-proof technology. 4.4. Black seed colour trait in quinoa – a mystery Four intra-specific crosses were performed taking black seeded D12377 as male parent with four different white seeded female parents as shown in Fig. 7 . All four crosses produced white coloured F 1 seeds. The F 1 seeds were grown and the F 1 plants produced black coloured F 2 seeds, thereby indicating toward dominance of the black seed colour (Fig. 7 ). Further, in order to understand the dominance/ recessive behaviour of seed colour, the black 76 F 2 seeds of two populations viz . Titicaca × D12377 and Chen281 × D12377 were grown to produce F 2:3 seeds. Assuming that the seed colour is the trait of seed itself, expected Mendelian ratio of black vs white seeds should be ~ 5:3. To confirm this, chi-squared test was performed which ruled out the significance of Mendelian 5:3 ratio (Table 3 ). Therefore, the possibility of dominance of black seed colour in quinoa germplasm materials investigated in this study was ruled out in this study. However, more robust and large-scale studies may help for an improved understanding of the dominance/recessive nature of black seed colour in quinoa. Further, in another experiment reciprocal crosses with white and black-seeded quinoa genotypes were made to better understand the black and white seed colour inheritance. Interestingly, these results revealed maternal and paternal seed colour trait expressions in F 1 and F 2 seeds respectively as presented in Fig. 8 . The paternal seed colour expression in F 2 seeds might be attributed to the pollen mediated gene transfer/expression/interaction in quinoa. The evidences generated in current study were inconclusive. Therefore, in conclusion, further in-depth genetic, cytological and molecular analyses are required to better understand the seed colour inheritance in quinoa. 4.5. Genetic improvement of quinoa with improved breeding methods and approaches Our study revealed the suitability of quinoa crossing approaches for diverse germplasms harbouring an array of floral diversity. In this study we have presented distinctive diagnostic traits that can help in improving efficiency of quinoa breeding, specifically differentiating characters of hermaphrodite flowers in some quinoa accessions. These distinctive traits enabled visual/ morphological identification of the true hybrids. In addition, we have observed the effectiveness of combined approach including both diagnostic and molecular markers in hybridity testing and genetic analysis that are important pre-requisites of a ‘breeding program’. The distinctive traits and floral arrangements of some accessions were so effective, that they facilitated “deliberate natural out-crossing” in quinoa, for example the black seed colour of accession, “D12377” or purple panicle colour of “Co 407”. Our findings suggest that large-scale identification of distinctive diagnostic characteristics in quinoa is urgently required. A feasible quinoa breeding scheme has been presented in Fig. 9 . The success of the proposed breeding scheme underlies parental stock with distinctive diagnostic characteristics, for example, black seed, purple panicle, coloured hermaphrodite flowers etc. Secondly, the cross ability due to floral size and shape of parental lines is a critical parameter which significantly affects the natural crossing. The parental stock lines with distinctive diagnostic characteristics could be planned in a specific arrangement to facilitate natural crossing followed by selection of putative F1s based on key adaptive traits such as vigor, color, grain yield or earliness. Thereafter, generation advance can be made by breeding selections for preferred traits in every generation till sufficient homozygosity is attained. These fixed lines can then be subjected to multilocation yield stability trails as part of varietal pipeline. During this process, fixed genotypes with unique diagnostic characters can be identified and added to the parental stock for the next cycle of breeding advancements. Similar breeding schemes can be formulated based on identified distinctive diagnostic characteristics and pursued for breeding suitable varieties. These efforts are crucial for the global expansion of quinoa, particularly in newer environments of Asia and Africas. 5. Conclusions The complex floral morphology of quinoa is the most important bottleneck in crossing and thereby its genetic improvement, which ultimately renders limited varietal diversity for varying crop ecologies in different parts of the world. The study presents the importance of the characterization of germplasm resources for the identification of the distinctive diagnostic traits that can be further utilized in overcoming crossing barriers. The study also validates the importance of ‘indel’ markers in quinoa breeding, particularly the marker-assisted hybridity testing. In this research, an interesting pattern was observed related to the black seed color of quinoa, which requires further in-depth analysis to better understand the genetics behind it. In a nutshell, quinoa improvement for newer environments can be followed in a sequential manner starting from crossing block definition, distinctive diagnostic trait identification for overcoming crossing barriers to marker-assisted hybridity testing, and breeding selection for ecology-specific desired traits. Declarations Acknowledgments The authors hereby duly acknowledge funding support provided by the International Center for Biosaline Agriculture, UAE. Authors are also thankful to all intellectual and non-technical inputs provided by researchers at ICBA and other institutions. Author contributions PV, RKS and HR designed experiments; PV and SVJ were responsible for crossing, PV, LM, HR and SA performed hybridity testing; SNR and MGK were responsible for data gathering; SS, PV, GPM and RKS interpreted the results; PV drafted the manuscript. All authors contributed to the article and approved the submitted version. Data availability statement The original contributions presented in the study are included in the article/Additional Material. Further inquiries can be directed to the corresponding author. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This research was conducted by institutional research fund of the International Center for Biosaline Agriculture, UAE. Ethics approval and consent to participate Not applicable. References Jacobsen SE (1997) Adaptation of quinoa ( Chenopodium quinoa ) to Northern European agriculture: studies on developmental pattern. Euphytica 96:41–48 Jacobsen SE (2003) The worldwide potential of quinoa ( Chenopodium quinoa Willd.). Food Rev Int 19:167–177 Jacobsen SE (2015) Adaptation and scope for quinoa in northern latitudes of Europe. In: FAO, CIRAD (eds) State of the art report on quinoa around the world in 2013. FAO, Rome, pp 436–446 Amin QA, Wani TA, Nahvi AI (2022) Quinoa is a gluten-free, high-nutrient and underutilized crop with a lot of processing possibilities. Agric Food E-Newsl 2:3 Anderson JA, Churchill GA, Autrique JE, Tanksley SD, Sorrells ME (1993) Optimizing parental selection for genetic linkage maps. Genome 36:181–186 Bazile D, Jacobsen SE, Verniau A (2016) The global expansion of quinoa: trends and limits. Front Plant Sci 7:622. https://doi.org/10.3389/fpls.2016.00622 Collard BCY, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86–93 Cota-Sanchez JH, Remarchuk K, Ubayasena K (2006) Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue. Plant Mol Biol Rep 24:161–167 Emrani N, Hasler M, Patiranage DSR, Maldonado N, Rey E, Jung C (2020) An efficient method to produce segregating populations in quinoa ( Chenopodium quinoa ). Plant Breed 139:1190–1200. https://doi.org/10.1111/pbr.12858 Fleming JE, Galwey NW (1995) Quinoa ( Chenopodium quinoa ). In: Williams JT (ed) Cereals and pseudocereals. Chapman and Hall, London, Underutilized Crops Series, vol 2, pp 3–83 James LEA (2009) Quinoa ( Chenopodium quinoa Willd.): composition, chemistry, nutritional, and functional properties. Adv Food Nutr Res 58:1–31 Koziol MJ (1991) Afrosimetric estimation of threshold saponin concentration for bitterness in quinoa ( Chenopodium quinoa Willd.). J Sci Food Agric 54:211–219 Murphy KM, Bazile D, Kellogg J, Rahmanian M (2016) Development of a worldwide consortium on evolutionary participatory breeding in quinoa. Front Plant Sci 7:608. https://doi.org/10.3389/fpls.2016.00608 Patirange DS, Rey E, Emrani N, Wellman G, Schmid K, Schmöckel SM et al (2022) Genome-wide association study in the pseudocereal quinoa reveals selection pattern typical for crops with a short breeding history. Elife 11:e66873. https://doi.org/10.7554/eLife.66873 Peterson A, Jacobsen SE, Bonifacio A, Murphy K (2015) A crossing method for quinoa. Sustainability 7:3230–3243. https://doi.org/10.3390/su7033230 Rahman H, Vikram P, Hu Y, Asthana S, Tanaji A, Suryanarayanan P et al (2024) Mining genomic regions associated with agronomic and biochemical traits in quinoa through GWAS. Sci Rep 14:19205. https://doi.org/10.1038/s41598-024-69992-2 Sagar P, Chatterjee S, Joshi DC, Tiwari JK (2025) Standardization of different techniques for the rapid production of F1 hybrids in Chenopodium quinoa . Euphytica 221:123. https://doi.org/10.1007/s10681-024-03456-7 Sha X (2013) Rice artificial hybridization for genetic analysis. Methods Mol Biol 956:1–12 Simmonds N (1971) The breeding system of Chenopodium quinoa I. Male sterility. Heredity 27:73–82 Singh D (2019) Quinoa ( Chenopodium quinoa Willd.). Scientific Publishers, Jodhpur Tabatabaei I, Alseekh S, Shahid M, Leniak E, Wagner M, Mahmoudi H et al (2022) The diversity of quinoa morphological traits and seed metabolic composition. Sci Data 9:323. https://doi.org/10.1038/s41597-022-01435-8 Ward S, Johnson D (1994) A recessive gene determining male sterility in quinoa. J Hered 85:231–233 Zhang T, Gu M, Liu Y, Lv Y, Zhou L et al (2017) Development of novel InDel markers and genetic diversity in Chenopodium quinoa through whole-genome re-sequencing. BMC Genomics 18:685. https://doi.org/10.1186/s12864-017-4003-2 Zurita-Silva A, Fuentes F, Zamora P, Jacobsen SE, Schwember AR (2014) Breeding quinoa ( Chenopodium quinoa Willd.): potential and perspectives. Mol Breed 34:13–30 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 15 May, 2026 Reviewers agreed at journal 14 May, 2026 Reviews received at journal 29 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 18 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor invited by journal 17 Apr, 2026 Editor assigned by journal 15 Apr, 2026 Submission checks completed at journal 15 Apr, 2026 First submitted to journal 11 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9391447","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":626866478,"identity":"b0aeae88-c8d4-4408-b65d-9ae074a8c98f","order_by":0,"name":"Prashant Vikram","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYFACxmYYy4AZRPKDiIQCErRISDaAtBjgtYYZVYvBATAbt3pzieRm44qabfb87M3bpAsq7OqMz69O/PDAgEGeX+wAVi2WMxKbE88cu504s+dYmfSMM8kSZjfebpYAOsxw5uwErFoMzhxsPtjAdjvB4EaO2W3eNmaglrMbQFoSDG7j0/Lvtr39/TdALf/qJYxnnN38A6+W443NiY1ttxk3SPAAtTQcljDg792G1xbL9sZmw8a+24kzzqSV/+Y5dlxyxg3ebRYJBhI4/WLOzP5YsuHbbXv+9sObjXlqqvn5+89uvvmjwkaeXxqHwzCFJMAqJbAqx6GF/wBO1aNgFIyCUTAyAQDmp2NfVQ/YAwAAAABJRU5ErkJggg==","orcid":"","institution":"International Center for Biosaline Agriculture","correspondingAuthor":true,"prefix":"","firstName":"Prashant","middleName":"","lastName":"Vikram","suffix":""},{"id":626866481,"identity":"580d803a-9c66-44dc-af60-2a75ad3a7b74","order_by":1,"name":"HIFZUR RAHMAN","email":"","orcid":"","institution":"International Center for Biosaline Agriculture","correspondingAuthor":false,"prefix":"","firstName":"HIFZUR","middleName":"","lastName":"RAHMAN","suffix":""},{"id":626866487,"identity":"3da8b293-1543-4866-a7df-7daec8f926ef","order_by":2,"name":"Lovely Mehta","email":"","orcid":"","institution":"International Center for Biosaline Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Lovely","middleName":"","lastName":"Mehta","suffix":""},{"id":626866488,"identity":"aa65efa6-7010-4279-bce0-ef2e2651e3cb","order_by":3,"name":"Sakshi Vidit Jain","email":"","orcid":"","institution":"International Center for Biosaline Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Sakshi","middleName":"Vidit","lastName":"Jain","suffix":""},{"id":626866490,"identity":"77e3db6c-88b3-4495-89ff-babbccb329fc","order_by":4,"name":"Sugandha Asthana","email":"","orcid":"","institution":"International Center for Biosaline Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Sugandha","middleName":"","lastName":"Asthana","suffix":""},{"id":626866491,"identity":"f1efb3a5-9cf6-4dcb-ae33-a0439eaf1841","order_by":5,"name":"Sowkat Nabi Rather","email":"","orcid":"","institution":"International Center for Biosaline Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Sowkat","middleName":"Nabi","lastName":"Rather","suffix":""},{"id":626866492,"identity":"13189374-4caf-42e0-8729-3013d7e4eda3","order_by":6,"name":"Murali Gumma Krishna","email":"","orcid":"","institution":"International Crops Research Institute for the Semi-Arid Tropics","correspondingAuthor":false,"prefix":"","firstName":"Murali","middleName":"Gumma","lastName":"Krishna","suffix":""},{"id":626866493,"identity":"aa348ae5-56bc-4038-979a-6175f3c880b1","order_by":7,"name":"Srinivasan Samineni","email":"","orcid":"","institution":"International Center for Biosaline Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Srinivasan","middleName":"","lastName":"Samineni","suffix":""},{"id":626866494,"identity":"5bc17be5-d1ca-416f-bdfc-958f6db21217","order_by":8,"name":"Gyan Prakash Mishra","email":"","orcid":"","institution":"Indian Agricultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Gyan","middleName":"Prakash","lastName":"Mishra","suffix":""},{"id":626866495,"identity":"9543b800-e87e-4835-ae06-95ee36d088f5","order_by":9,"name":"Rakesh Kumar Singh","email":"","orcid":"","institution":"International Center for Biosaline Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Rakesh","middleName":"Kumar","lastName":"Singh","suffix":""}],"badges":[],"createdAt":"2026-04-12 03:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9391447/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9391447/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107909919,"identity":"40e1cc81-aabe-4834-ac2f-df80ddaf16e6","added_by":"auto","created_at":"2026-04-27 13:12:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":134454,"visible":true,"origin":"","legend":"\u003cp\u003eFigure presenting the arrangement of five different quinoa genotypes with distinctive diagnostic characteristics. Orientation of all the five plants was kept same in all pots.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/d61525a8ffd47aeba6877b15.jpeg"},{"id":107909925,"identity":"fe74eaba-c527-40f8-992c-46ff24b2110e","added_by":"auto","created_at":"2026-04-27 13:12:08","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81266,"visible":true,"origin":"","legend":"\u003cp\u003eDendrogram presenting diversity pattern of the crossing block accessions.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/72ac84fcc6183458065bd067.jpeg"},{"id":107909930,"identity":"91fcc129-7b32-4a95-86f2-89f247a0e20d","added_by":"auto","created_at":"2026-04-27 13:12:10","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":157194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A):\u003c/strong\u003e Hybridity testing of the cross between well adapted variety, ‘Titicaca’ and saponin free line ‘D12401’; \u003cstrong\u003e(B):\u003c/strong\u003e Hybridity testing of the cross between well adapted variety, ‘Titicaca’ and saponin free black seeded line ‘D12377’; \u003cstrong\u003e(C):\u003c/strong\u003e Hybridity testing of the cross between Chenopodium giganteum (Ames 19046) and Chenopodium quinoa (D12406)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/6440eca5af68fbffe15dce42.jpeg"},{"id":107909894,"identity":"d9de9916-8058-4812-a036-d04a5a902e5d","added_by":"auto","created_at":"2026-04-27 13:12:00","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":257890,"visible":true,"origin":"","legend":"\u003cp\u003eFigure presenting the natural crossing scheme using black seeded quinoa enabling hybrid identification through seed colour of progenies\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/e3854b604efa2bc4a8e74337.jpeg"},{"id":107909934,"identity":"54dedaa8-a6b8-4013-bccd-1a2f434ca963","added_by":"auto","created_at":"2026-04-27 13:12:11","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":162108,"visible":true,"origin":"","legend":"\u003cp\u003ePink colored hermaphrodite flowers identified in quinoa accession- D12401\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/8fe95d7fbfafac48ed19e61d.jpeg"},{"id":107909918,"identity":"4a5ed979-f668-4f4a-8c35-a5a2cdd26f32","added_by":"auto","created_at":"2026-04-27 13:12:07","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":321664,"visible":true,"origin":"","legend":"\u003cp\u003eFigure presenting morphological identification of true crosses in quinoa having diagnostic characteristics. Among 5 parents that underwent natural crossing, Co 407 was white seeded - purple panicle plant, whereas D-12377 was black seeded –green panicle plant. The naturally crossed panicle presented here had purple panicle harbouring black seed confirming cross between Co 407 and D12377. Yellow arrows pointing the black seed formation\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/553964f684dca57b837c09ca.jpeg"},{"id":107909929,"identity":"9a04d18f-9170-4fd7-a9c8-2e8979d107bf","added_by":"auto","created_at":"2026-04-27 13:12:09","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":806485,"visible":true,"origin":"","legend":"\u003cp\u003eFigure presenting the dominance of black seed colour in quinoa. Black seeded parent was crossed with four different white seeded genotypes and F2 from all four crosses were black.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/648e8b24d20a2b6d979e32cd.jpeg"},{"id":107909926,"identity":"52f85a04-0b72-4a18-827f-7adecd8165da","added_by":"auto","created_at":"2026-04-27 13:12:09","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":147760,"visible":true,"origin":"","legend":"\u003cp\u003eFigure presenting the black – white seed colour variation in reciprocal crosses of quinoa\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/02a564cdb958218076294515.jpeg"},{"id":107909921,"identity":"3ecb02b7-e8bd-409d-978f-ff9838a4129e","added_by":"auto","created_at":"2026-04-27 13:12:07","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":213946,"visible":true,"origin":"","legend":"\u003cp\u003eFlow chart proposing a fast-track quinoa breeding pipeline strategy through identifying and utilizing parental stocks with diagnostic characteristics\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/5817364838ef94d51a35a336.jpeg"},{"id":107910018,"identity":"bc84dbc2-f993-406d-9774-475cdfc2f8ec","added_by":"auto","created_at":"2026-04-27 13:12:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2679039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/83fc0eb4-6004-4c1c-be93-f7ef85aed425.pdf"},{"id":107909924,"identity":"8d693cab-9b5b-4a14-84aa-643949d1c753","added_by":"auto","created_at":"2026-04-27 13:12:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1072226,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9391447/v1/579e2aac7c4ad4f2728c0c7d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eStrategic Breeding Approaches: Harnessing Molecular and Phenotypic Markers to Overcome Quinoa's Flower Morphology Bottlenecks\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eQuinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e) is one of the oldest crops of the American continent, believed to have originated and cultivated in the Andean region, mainly in Peru, Bolivia, and Chile. Quinoa can be considered a new gift of the \u0026lsquo;new world\u0026rsquo; to the \u0026lsquo;old world\u0026rsquo;, like the potato and tomato. Quinoa has earned special attention worldwide due to its exceptional nutritional quality, health benefits and ability to adapt to marginal environments (Jacobsen, 1997, 2003, 2015).\u003c/p\u003e \u003cp\u003eQuinoa, a pseudocereal due to its grain structure, is an annual dicotyledonous species that belongs to the family \u003cem\u003eAmaranthaceae\u003c/em\u003e (formerly \u003cem\u003eChenopodiaceae\u003c/em\u003e). Quinoa is exemplary for amino acid composition and offers a remarkable balance of oil (4\u0026ndash;9%), protein (averaging 16%, with high nutritional value), and carbohydrates (64%) (James 2009). It can be used to make flour in the same way as cereals due to its high starch content (51\u0026ndash;61%). Quinoa grain is free of gluten, which has facilitated the creation of various products for people with celiac disease (i.e., those who are allergic to gluten) (Singh 2019, Amin et al. 2022).\u003c/p\u003e \u003cp\u003eQuinoa is one of the oldest crops but its limited spread happened in the past few decades (Bazile et al. 2016). The major reason for the limited spread is primitive breeding efforts, mostly reported from Bolivia and Peru (Zurita-Silva et al. 2014). Although some recent breeding studies have reported the development of high-yielding varieties adapted to temperate regions and high latitudes of Europe, North America, and China, most of these were limited to mutation breeding or mass selection (Murphy et al. 2016, Patirange et al. 2022). Many Asian and African regions are continuously making efforts to increase the genetic biodiversity of the quinoa cropping system by hybridization to develop high-yielding, sweet and improved varieties, with limited success (Murphy et al. 2016).\u003c/p\u003e \u003cp\u003eThe biggest challenge in large-scale quinoa breeding is due to its very small florets and the presence of both hermaphrodite and female flowers (out-crossing 0.5% to 17%) on the same panicle (Murphy et al. 2016). Due to the complex arrangement of the hermaphrodite and pistillate (female) flowers, small size of flowers and fast progression of flowering within infloresence it becomes very difficult to perform hand emasculation efficiently (Paterson et al. 2015). Hot water treatment is another method followed for emasculation in which hot water (~\u0026thinsp;45\u0026deg;C) application is done on the inflorescence in a way to inactivate the pollens while keeping ovaries and stigma safe, however, this method is also reported to damage the inflorescence (Fleming et al. 1995, Sha 2013). Though it is time-consuming, imparting male sterility can be an alternative method of creating hybrids. Some male sterility sources have been identified in quinoa including cytoplasmic male sterility (CMS) germplasms along with their restorer genes (Simmonds 1971, Ward and Johnson 1994). It is important to note that heterosis has not been reported in quinoa so far, so, application of CMS system might not be very feasible option, hence not pursued further at commercial scale.\u003c/p\u003e \u003cp\u003eQuinoa is also known for its highly variable outcrossing rates across different accessions (Murphy et al. 2016) that can be utilized for enhancing the quinoa breeding program efficiency. Identification of the true F\u003csub\u003e1\u003c/sub\u003es is another important concern in quinoa breeding. Broadly, there are two strategies implied for the identification of F\u003csub\u003e1\u003c/sub\u003e plants in quinoa i.e. using morphological and/or molecular markers. Traits such as axillary pigmentation, plant colour, seed colour and inflorescence can be used as diagnostic markers provided the male parent (pollinator) is homozygous for the dominant alleles (Paterson et al. 2015). On the other hand, molecular marker use in hybridity testing requires polymorphic markers between the two parents of the F\u003csub\u003e1\u003c/sub\u003e plants.\u003c/p\u003e \u003cp\u003eIn the current study, efforts have been made to employ different crossing strategies to establish a robust quinoa breeding platform specifically for the newer environments including Asia/ Middle East.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. The quinoa crossing block\u003c/h2\u003e \u003cp\u003eQuinoa breeding work was initiated using gene bank accessions stored in the International Centre for Biosaline Agriculture (ICBA), Dubai, UAE, in December 2020. A subset from these accessions was subjected to saponin estimation analysis and reported recently (Tabatabei et al. 2022). Low saponin accessions were shortlisted from the report of Tabatabaei et al. 2021, and, validated through reanalysis following afrosimetric method (Koziol 1991). Seven parents were found to be suitable donors for low saponin parents (Ames 19046, D12401, D12406, D 11912, D12377, Chen281 and Chen254). Based on visual observations, six other donors (Co 407, D12047, ICBA-Q5, BO 51, Titicaca and ICBA-Q3) were selected for grain yield and adaptive characteristics (Supplementary Figs.\u0026nbsp;1a-b). ICBA-Q5 and ICBA-Q3 are two lines selected at ICBA and were nominated as varieties in different countries (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.biosaline.org/news/2020-03-14-7046\u003c/span\u003e\u003cspan address=\"https://www.biosaline.org/news/2020-03-14-7046\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Titicaca is a widespread variety of Europe; Co 407 is a variety from Colorado State University, USA, developed by mutation. D12047 and BO 51 were identified as high-yielding accessions during field experiments for the GWAS study at ICBA fields in the 2020-21 trial (Rahman et al. 2024).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. DNA extraction and PCR amplification\u003c/h2\u003e \u003cp\u003eGenetic analyses were performed for the characterization of crossing blocks and testing of hybridity. DNA of the crossing block lines and F\u003csub\u003e1\u003c/sub\u003e plants were extracted following a modified CTAB method explained by (Cota-Sanchez et al. 2006). The InDel markers reported by (Zhang et al. 2017) were synthesized, and a parental polymorphism survey was carried out on the parental DNA. DNA was amplified using protocol explained by (Collar and Mackill, 2009). Reaction mixture of 20 \u0026micro;L included 10 \u0026micro;L of master (2X FIREPol\u0026reg; master mix), 0.5 \u0026micro;L of (0.1 \u0026micro;M) for each primer, and 1 \u0026micro;L of genomic DNA (50 ng/\u0026micro;L). The final volume was obtained using sterile distilled water. The PCR reaction was performed using a thermal profile with pre-denaturing for 5 min at 95\u0026deg;C, followed by 40 cycles at 94\u0026deg;C for 40 s, annealing for 40 s, and extension at 72\u0026deg;C for 30 sec, with a final extension time of 5 min at 72\u0026deg;C. PCR products were separated on a 3.0% agarose gel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Genetic analysis\u003c/h2\u003e \u003cp\u003eData were scored as (1) for presence and (0) for absence for each sample under investigation. The polymorphism information content (PIC) was calculated according to the formula: PIC\u0026thinsp;=\u0026thinsp;1\u0026thinsp;\u0026minus;\u0026thinsp;Σ\u003cem\u003epi\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e where pi is the frequency of the \u003cem\u003ei\u003c/em\u003eth allele of the locus in eight genotypes (Anderson et al. 1993).The data was analysed with SIMQUAL program of NTSYS-pc (Version 2.02), and similarities between accessions were estimated using the Jaccard\u0026rsquo;s coefficient calculated as J\u0026thinsp;=\u0026thinsp;A / (N - D), where A is the number of positive matches (that is, presence of band in both samples), D is the number of negative matches (that is, absence of band in both samples) and N is the total sample size including both the number of matches and unmatched. A dendrogram was created from the resultant similarity matrices using the UPGMA (Unweighted Pair Group Method with Arithmetic mean) method following the SAHN function of NTSYS-pc (Version 2.02).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Crossing and population advancement\u003c/h2\u003e \u003cp\u003eMaking crosses in quinoa is challenging because of its gyno-monoecious flowers and high resemblance of the hermaphrodite and female ones. Three different approaches were followed for making crosses by: (1) removing hermaphrodite flowers from the female parents, (2) forced synchronization of flowering and (3) natural crossing.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Crossing by removing hermaphrodite flowers\u003c/h2\u003e \u003cp\u003eAll 13 parents were sown in staggered planting mode in ICBA\u0026rsquo;s green house during 5th December 2020 to 31st January 2021 at 15 days interval. At the peak flowering stage, hermaphrodite flowers were removed from the inflorescence leaving only female flowers on female parents and clubbed with that of male parent (in which hermaphrodite flowers were not removed) and covered with a glassine bag as illustrated in Supplementary Figs.\u0026nbsp;2 (a) and (b). Hermaphrodite flowers were observed and identified using magnifying glasses (Supplementary Fig.\u0026nbsp;2c). One of the 13 parents was \u003cem\u003eChenopodium giganteum\u003c/em\u003e (Ames 19046) which was crossed with four \u003cem\u003eChenopodium quinoa\u003c/em\u003e accessions viz. D-12406, ICBA-Q3, Chen-254 and D-12377 (Supplementary Fig.\u0026nbsp;2b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Forced synchronization of flowering\u003c/h2\u003e \u003cp\u003eFive genotypes were identified for crossing through controlled flowering to force synchronization of anthesis: 1. ICBA-Q5, 2. D12401, 3. D12406, 4. Co 407 and 5. D12377. Selection of these five genotypes was done based on their unique characteristics including D12401: purple colour hermaphrodite flower, D12406: similar to D12401 but white colour hermaphrodite flowers, D12377: black seeded, Co 407: purple panicle and ICBA-Q5: Very early maturing. These five genotypes were sown in a clockwise fashion in the pot starting from #1 to #5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThey were first allowed to grow vegetatively, however, during flowering stage, early flowering panicles were cut with scissors in order to synchronize their flowering with other lines (Supplementary Fig.\u0026nbsp;2d). Quinoa plant has characteristic of continuously flowering for several weeks. This characteristic was exploited in this approach. We continued synchronization until when majority of the five plants flowered synchronously. After manual removal of panicles from the top, plants became usually short heighted and tender so that it became easy to tie (loosely) all of them and cover with a big glassine bag. Later on, each of the five lines were harvested from different pots and bulked. Seeds of individual five lines were sown in the next season in single plots so that crosses individuals can be visualized with the help of their parental distinctive diagnostic characteristics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Natural crossing strategy through no emasculation method\u003c/h2\u003e \u003cp\u003eIn another approach, the pots with five genotypes (D12401, D12406, D12377, Co 407 \u0026amp; ICBA-Q5) with distinctive morphological characteristics as explained in the above section were kept just in front of the cooling pad of greenhouse so that slowly blowing wind from fan can facilitate movement of pollen from one genotype to another (Supplementary Fig.\u0026nbsp;2e). This way natural out crossing was facilitated. Each of the five genotypes were harvested from all pots and bulked for the next season sowing. As mentioned above, each line was harvested individually from different pots and formed five different bulks. Seeds of individual bulks were grown in the next season in individual plots to visualize probable crosses with the help of distinctive diagnostic characteristics. This method presents a slight modification in the \u0026acute;no emasculation method\u0026acute; as reported by (Emrani et al. 2020).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Saponin estimation of the crossing block\u003c/h2\u003e \u003cp\u003eBased on the saponin estimation analysis according to using afrosimetric method (Michael J Koziol 1991) parents for quinoa crossing block were declared. A total of seven genotypes were found suitable to use as saponin free donor parents (Ames 19046, D12401, D12406, D 11912, D12377, Chen281 and Chen254), whereas six genotypes were used as parents for high grain yield and adaptive characteristics (Co 407, D12047, ICBA-Q5, BO 51, Titicaca and ICBA-Q3) based on in house experiments (ICBA Unpublished). Results are depicted in Supplementary Figs.\u0026nbsp;1(a) and (b). Unique characteristics of all the parents are delineated in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSalient characteristics of the parents used for quinoa crossing block\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKey Trait\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRemarks\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmes-19046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaponin free-Super late\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePreferred for Forage, different grain and plant type.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChen-254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaponin free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChen-281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaponin free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-11912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaponin free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-12377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaponin free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis is a heterogenous line producing two types of seed (brown \u0026amp; black), its f loral biology and flowering pattern need to be studied.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-12401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaponin free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium, this line has diagnostic hermaphrodite flower in certain environmental condition (pink color hermaphrodite flower, if grown in open light condition).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-12406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaponin free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBO-51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYield \u0026amp; Adaptation Traits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCo-407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYield \u0026amp; Adaptation Traits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-12047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYield \u0026amp; Adaptation Traits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis might be a segregating line, need not to use in breeding further.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICBA-Q3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYield \u0026amp; Adaptation Traits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICBA-Q5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYield \u0026amp; Adaptation Traits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTiticaca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYield \u0026amp; Adaptation Traits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVery Early\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Genetic profiling of parents in crossing block\u003c/h2\u003e \u003cp\u003eOut of 36 indel primers studied 25 indels showed polymorphism among different accessions of crossing block. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents relationship among different crossing block accessions. Dendrogram clearly reveals that \u003cem\u003eChenopodium giganteum\u003c/em\u003e (Ames 19046) is distantly related with \u003cem\u003eChenopodium quinoa\u003c/em\u003e genotypes of crossing block. Broadly, there were two groups i.e. GP-I and GP-II; GP-I had three saponin free lines Chen254, D12406 and D 11912, whereas GP-II included ten diverse accessions including Ames 19046, D12401, D12377, Chen281, Co 407, D12047, ICBA-Q5, BO 51, Titicaca and ICBA-Q3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Efficiency of three crossing methods\u003c/h2\u003e \u003cp\u003eThe hybridity testing of 15 cross combinations ascertained through seeds obtained from female flowers after removing hermaphrodite flowers from female parents, crossing efficiency was found in range of 5 to 71% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). In the interspecific crosses of \u0026lsquo;\u003cem\u003eChenopodium quinoa \u0026times; Chenopodium giganteum\u003c/em\u003e\u0026rsquo; this Figure ranged 5\u0026ndash;26%, whereas, among \u0026lsquo;\u003cem\u003eChenopodium quinoa \u0026times; Chenopodium quinoa\u003c/em\u003e\u0026rsquo; crosses it was 10\u0026ndash;71%. While making interspecific crosses \u003cem\u003eChenopodium giganteum\u003c/em\u003e was used as male parent because of its floral arrangement and multiple hanging branches. Among four interspecific crosses, \u0026lsquo;ICBA-Q3 \u0026times; Ames-19046\u0026rsquo; did not produced viable seed, whereas, other three successfully produced viable seeds. Maximum number of true hybrids were observed in \u0026lsquo;D-12401 \u0026times; BO-51\u0026rsquo; cross i.e. 15 out of 21 (71%), and minimum crossing efficiency was revealed in \u0026lsquo;D-12401 \u0026times; ICBA-Q3\u0026rsquo; as presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTable presenting the bi-parental crosses confirmed with Indel-based markers, crossing efficiency has been presented with respect to individuals tested in the laboratory.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCross Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRef#\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCross Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. of true hybrids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal number of seed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCrossing Efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCrossing Date\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cem\u003eChenopodium quinoa \u0026times; Chenopodium giganteum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12406\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; Ames-19046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e03/02/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC-18*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eICBA-Q3\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; Ames-19046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e03/01/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChen-254\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; Ames-19046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e03/08/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12377\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; Ames-19046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e03/08/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"10\" rowspan=\"11\"\u003e \u003cp\u003e\u003cem\u003eChenopodium quinoa \u0026times; Chenopodium quinoa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12401\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; ICBA-Q3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14/02/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAC-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eICBA-Q5\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; D-12377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e04/07/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eICBA-Q5\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; ICBA-Q3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24/02/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAC-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBO-51\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; D-12377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e04/11/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB-37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12377\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; ICBA-Q5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e03/14/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB-42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12406\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; ICBA-Q5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e03/15/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12377\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; Chen-281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e04/07/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTiticaca\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; D-12377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e02/08/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB-44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12406\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; ICBA-Q3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e03/01/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB-25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12401\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; ICBA-Q5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e04/14/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB-24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD-12401\u0026nbsp;\u0026nbsp; \u0026times;\u0026nbsp;\u0026nbsp; BO-51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e04/14/2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong five parents used for natural crossing, only D12377 was black seeded parent, and this trait was used for determining the efficiency of natural crossing. There were total of 10 pots each having five parents for natural crossing purpose including D12377 as one of them. All the D12377 plants (i.e. 10 plants, one from each pot) were harvested and seed was bulked. A total of 1000 seed were sown to get 823 plants from naturally crossed black seeds (produced with D12377 as female parent). Among 823 plants, 205 and 618 produced white and black seeds respectively. As mentioned above, the 1000 seeds sown from 10 different supposedly, naturally crossed D12377 plants (i.e. we are not sure which of them underwent natural crossing). In way to analyse the crossing efficiency, we here assume that the 618 plants producing black seeds came from selfed black seeded D12377 and vice-versa for the rest 205 white seed producing plants. Therefore, with this assumption (618 black seeded plants are from selfed D12377 parent), we can inference that 205/618 \u0026times; 100\u0026thinsp;~\u0026thinsp;25% would be the minimum crossing efficiency. It is important to note here that this efficiency was achieved under a specific situation i.e. with five plants sown in a pot and kept together in very closed vicinity and 618 black seeded plants were assumed to be derived from selfed plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eManual observation based on distinctive diagnostic characteristics of the five parents subjected to forced flowering synchronization, crossing efficiency was observed in range from 1.9% in D12406 (5/263) to 7.7% in ICBA-Q5 (6/78). In D12401 (16/410) and D12377 (6/154) efficiency was approximately 4% (data not presented).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Inter-specific cross compatibility\u003c/h2\u003e \u003cp\u003eAmong four inter-specific crosses attempted in our study with \u003cem\u003eChenopodium giganteum\u003c/em\u003e (Ames 19046) as common male parent and Chen254, D12406, D12377 and ICBA-Q3 as females (Supplementary Figs.\u0026nbsp;3a-b). F\u003csub\u003e1\u003c/sub\u003e seeds of one cross (i.e. Ames 19046 \u0026times; ICBA-Q3) did not produced F\u003csub\u003e2\u003c/sub\u003e seeds (Supplementary Fig.\u0026nbsp;3c). The other three crosses, Ames 19046 \u0026times; Chen254, Ames 19046 \u0026times; D12406 and Ames 19046 \u0026times; D12377 successfully produced F\u003csub\u003e2\u003c/sub\u003e seeds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Morphological marker identification\u003c/h2\u003e \u003cp\u003eWith respect to arrangement of hermaphrodite and female flowers, two variants were observed that could serve as morphological markers. The low saponin parent D12401 was identified most suitable for crossing because of its pink coloured hermaphrodite flowers which were clearly distinct from the female ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similarly, in one of the high saponin line D12047, hermaphrodite and female flowers were arranged in somewhat conical fashion, in which hermaphrodite flower was placed on the tip and female ones on side (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e derived from cross of D12377 \u0026times; ICBA-Q5 was identified as a distinctive diagnostic panicle colour as illustrated in (Supplementary Fig.\u0026nbsp;5). In the late maturity stage, panicle colour of D12377, ICBA-Q5 and F\u003csub\u003e1\u003c/sub\u003e were clearly distinct. Similarly, in the natural crossing scheme, five characteristically distinct genotypes (Co 407, D12401, D12406, D12377 and ICBA-Q5), were harvested and individually bulked.\u003c/p\u003e \u003cp\u003eCharacteristics of D12377 are green panicle and black seed colour. Another parent Co 407 has purple coloured panicle which produced white seeds. Therefore, if a F\u003csub\u003e1\u003c/sub\u003e plant derived from the cross of these two accessions might inherit at least one trait from each parent - D12377 and Co 407. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e presents a panicle of F1 plant in which the panicle colour is purple (similar to C0 407) producing black seed (trait from D12377). This indicates that the F\u003csub\u003e1\u003c/sub\u003e was produced from pollination of female flower of Co 407 by hermaphrodite flowers of D12377 or vice-versa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Seed colour observations in quinoa\u003c/h2\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e seeds derived from cross of five white seed (female) with one black seed parent (male), were all white i.e. female parent type. The F\u003csub\u003e2\u003c/sub\u003e seeds produced on all these five F\u003csub\u003e1\u003c/sub\u003es were found black coloured. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e presents the seed colour of parents (Titicaca, ICBA-Q5, ICBA-Q3, BO 51 and D12377) and F\u003csub\u003e2\u003c/sub\u003e seeds. Seeds of fifth cross of Chen281 (white seeded) \u0026times; D12377 showed similar pattern (less seeds were produced and advanced immediately, picture couldn\u0026rsquo;t be taken). These results indicated toward dominance of the black seed colour over white in quinoa. However, to better understand this pattern reciprocal crosses were made using white seeded female (ICBA-Q5) with black seeded male (D12377) and vice-versa. In both cases F\u003csub\u003e1\u003c/sub\u003e seeds tend to be maternal type which clearly revealed maternal inheritance, not the dominance of any seed coat colour (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Therefore, existence of white coloured F\u003csub\u003e2\u003c/sub\u003e seeds on F\u003csub\u003e1\u003c/sub\u003e plants made from the cross of white seeded male (ICBA-Q5) with black seeded female (D12377) rules out any conclusion on dominance of seed colour in quinoa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther, 76 F\u003csub\u003e2\u003c/sub\u003e seeds of each of the two populations Titicaca \u0026times; D12377 and Chen281 \u0026times; D12377 were advanced and colour of F\u003csub\u003e3\u003c/sub\u003e seeds were observed to better understand segregation pattern. The 76 F\u003csub\u003e2\u003c/sub\u003e plants of Titicaca \u0026times; D12377 produced 53 black and 23 white seeds in the ratio of 2.3:1. Similarly, 76 F\u003csub\u003e2\u003c/sub\u003e plants of Chen281 \u0026times; D12377 produced 51 black and 25 white seeds in the ratio of 2.07:1. Chi squared test performed in both crosses ruled out the significance of Mendelian monohybrid ratio of 5:3 in F3 generation indicating lack of complete dominance of black seed colour in quinoa (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTable presenting the chi-squared test to test \u0026ldquo;the goodness of fit\u0026rdquo; with the null hypothesis (H\u003csub\u003e0\u003c/sub\u003e): Ratio of Black and White seeds in F3 generation were 5:3. The F\u003csub\u003e2:3\u003c/sub\u003e seeds were produced by two populations i.e. \u0026lsquo;Titicaca \u0026times; D-12377\u0026rsquo; and \u0026lsquo;Chen 281 \u0026times; D-12377\u0026rsquo;\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTiticaca \u0026times; D-12377\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlack\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChen 281 \u0026times; D-12377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eChi-Square calculation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.N.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eObserved (O)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExpected (E)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eO-E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(O-E)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(O-E)\u003csup\u003e2\u003c/sup\u003e/E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.63684\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.0614\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.25789\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.42982\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eChi-Squared value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe chi-Squared value at α\u0026thinsp;=\u0026thinsp;0.05 with degree of freedom\u0026thinsp;=\u0026thinsp;1, the chi-squared value was 3.841. The calculated chi-Squared value was 2.38 which was lesser than 3.841 with 5% significance level. Therefore, we can reject the null hypothesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eImportance of quinoa outside its domestication centre i.e. Andes with ensured irrigation has been recognised in past few years. Quinoa can also be introduced/ scaled up in different parts of Asia and Africa by transforming the existing cropping systems and developing suitable varieties that can perform well under specific environments. Quinoa breeding work to develop the improved varieties is being undertaken at ICBA, UAE successfully.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Inter-specific crosses in \u003cem\u003eChenopodium species\u003c/em\u003e: Opportunity of exploiting variation\u003c/h2\u003e \u003cp\u003eDefining a crossing block of diverse genotypes is the first step in establishing a breeding program. Low saponin, early maturity and high yield were selected as the three most important traits required for the adaptation of quinoa in newer environments. Thirteen parents in crossing blocks comprised of six \u003cem\u003eChenopodum quinoa\u003c/em\u003e and one \u003cem\u003eChenopodum giganteum\u003c/em\u003e accessions with saponin free/ very low saponin content along with six other high yielding varieties/ genotypes (Supplementary Figs.\u0026nbsp;1a-b). Diversity pattern of these 13 genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) based on similarity index could be the best way to choose potential parents. Interestingly, F\u003csub\u003e1\u003c/sub\u003e plants from three interspecific crosses made with \u0026lsquo;saponin free \u003cem\u003eChenopodum giganteum\u003c/em\u003e\u0026rsquo; with \u0026lsquo;saponin free \u003cem\u003eChenopodum quinoa\u003c/em\u003e\u0026rsquo; successfully produced F\u003csub\u003e2\u003c/sub\u003e seeds, whereas F\u003csub\u003e1\u003c/sub\u003e of \u0026lsquo;saponin free \u003cem\u003eChenopodum giganteum\u003c/em\u003e\u0026rsquo; \u0026times; \u0026lsquo;high saponin \u003cem\u003eChenopodum quinoa\u003c/em\u003e\u0026rsquo; did not produced F\u003csub\u003e2\u003c/sub\u003e seeds.\u003c/p\u003e \u003cp\u003eNoticeably, the plant type of F\u003csub\u003e1\u003c/sub\u003es developed from inter-specific crosses (\u003cem\u003eChenopodum quinoa\u003c/em\u003e \u0026times; \u003cem\u003eChenopodum giganteum\u003c/em\u003e) resembled respective \u003cem\u003eChenopodum quinoa\u003c/em\u003e parent (D12406, D12377 and Chen254), and successfully produced the F\u003csub\u003e2\u003c/sub\u003e seeds. Conversely, plant type of F\u003csub\u003e1\u003c/sub\u003e developed from ICBA-Q3 (\u003cem\u003eChenopodum quinoa\u003c/em\u003e) \u0026times; Ames 19046 (\u003cem\u003eChenopodum giganteum\u003c/em\u003e) resembled \u003cem\u003eChenopodum giganteum\u003c/em\u003e parent (Ames 19046) and did not produce the F\u003csub\u003e2\u003c/sub\u003e seeds (Supplementary Fig.\u0026nbsp;3c). These observations clearly suggest that inter-specific crosses with \u003cem\u003eChenopodum quinoa\u003c/em\u003e with other \u003cem\u003eChenopodum spp.\u003c/em\u003e can be exploited for the desired genetic advance. The inter-specific cross compatibility and heterosis analyses can be performed at a large scale for identification of suitable genetic variants. In order to ensure adaptability of \u003cem\u003eChenopodum quinoa\u003c/em\u003e in newer environments, genetic variations created from inter-specific crosses can play a major role.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Role of distinctive phenotypic traits as diagnostic markers in natural crossing\u003c/h2\u003e \u003cp\u003eAmong 13 crossing block accessions, two distinctive diagnostic floral arrangements were observed that can greatly help in the manual crossing process. The pink-coloured hermaphrodite flowers with green-coloured female ones in quinoa genotype D12401 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and the positioning of hermaphrodite flowers on the top of a cone, while female ones on sides in D12047 (Supplementary Fig.\u0026nbsp;4) render them very easily discriminated from female flowers. These flowers can, therefore, be easily removed or destroyed and female ones can be pollinated with other accessions.\u003c/p\u003e \u003cp\u003eThe black seed colour was observed as another diagnostic characteristic that can be potentially used in natural crossing. Five quinoa genotypes underwent natural crossing (or out-crossing), as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and were harvested individually and grown in separate plots in the next generation i.e. one genotype in one plot. True hybrids were identified successfully with the help of seed colour of F\u003csub\u003e1\u003c/sub\u003e seeds. If black-seeded plants produced white seeds or vice-versa were declared as true hybrids. Similarly, the purple panicle colour of Co 407 was used as a diagnostic characteristic while crossing quinoa genotypes naturally. However, in every breeding program, hybridity testing using polymorphic markers is always advisable to employ as fail-safe technology.\u003c/p\u003e \u003cp\u003eThe probability of identifying the desired genetic variant increases by enhancing the number of crosses and population sizes. Therefore, a feasible crossing methodology ensuring a large number of crosses with minimal effort should help quinoa breeding greatly. The efficiency of three different crossing methods indicated the importance of quinoa germplasms with distinctive diagnostic characteristics, such as black seed colour and purple panicle. It was very interesting to note here that the outcrossing efficiency of the black-seeded quinoa line, D12377 was observed to be 25%, assuming that all other black seeds were selfed. Out of 823 putative F\u003csub\u003e1\u003c/sub\u003e plants taking D12377 as female parent (black seeded), 205 produced white seeds and rest 618 yielded black seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We did not observe any plant producing both black and white seed. The forced flowering synchronization method attempted in this study did not provide encouraging results, possibly because of trimming large number of flowers for synchronization. Manual crossing through removing hermaphrodite flowers, revealed a wide range of success rate indicating the role of varying floral arrangements among quinoa germplasm accessions. In nutshell, a large-scale effort in identification of distinctive diagnostic traits and floral biology in quinoa can help greatly in creating desired genetic variants that may further help in its wider adaptation in newer environments of Africa and Asia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Marker assisted breeding: Tool that can handle crossing efficiency issue in quinoa\u003c/h2\u003e \u003cp\u003eIt is a well-known fact that due to gyno monoecious nature of flowers in quinoa, manual emasculations become difficult and less efficient. Therefore, efforts have been made in past to emasculate using different approaches including manual emasculation and hot water treatments (Emrani et al. 2020, Peterson et al. 2015, Sagar et al. 2025). However, establishing breeding pipeline and making large-scale efforts would require feasibly applicable alternatives. In this study we have presented that hybridity testing of the putative crosses through mechanical emasculation (like removal of hermaphrodite flowers) as fool proof backup strategy using abundantly available indel polymorphic markers in quinoa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, the bottleneck of low efficiency of manual crossing in quinoa can be effectively addressed by using marker assisted breeding approaches. The aforesaid methods of crossing could be used to fast-track breeding efforts in difficult crop like quinoa, in conjunction with hybridity testing using indel markers as fool-proof technology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Black seed colour trait in quinoa \u0026ndash; a mystery\u003c/h2\u003e \u003cp\u003eFour intra-specific crosses were performed taking black seeded D12377 as male parent with four different white seeded female parents as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. All four crosses produced white coloured F\u003csub\u003e1\u003c/sub\u003e seeds. The F\u003csub\u003e1\u003c/sub\u003e seeds were grown and the F\u003csub\u003e1\u003c/sub\u003e plants produced black coloured F\u003csub\u003e2\u003c/sub\u003e seeds, thereby indicating toward dominance of the black seed colour (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Further, in order to understand the dominance/ recessive behaviour of seed colour, the black 76 F\u003csub\u003e2\u003c/sub\u003e seeds of two populations \u003cem\u003eviz\u003c/em\u003e. Titicaca \u0026times; D12377 and Chen281 \u0026times; D12377 were grown to produce F\u003csub\u003e2:3\u003c/sub\u003e seeds. Assuming that the seed colour is the trait of seed itself, expected Mendelian ratio of black vs white seeds should be ~\u0026thinsp;5:3. To confirm this, chi-squared test was performed which ruled out the significance of Mendelian 5:3 ratio (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, the possibility of dominance of black seed colour in quinoa germplasm materials investigated in this study was ruled out in this study. However, more robust and large-scale studies may help for an improved understanding of the dominance/recessive nature of black seed colour in quinoa.\u003c/p\u003e \u003cp\u003eFurther, in another experiment reciprocal crosses with white and black-seeded quinoa genotypes were made to better understand the black and white seed colour inheritance. Interestingly, these results revealed maternal and paternal seed colour trait expressions in F\u003csub\u003e1\u003c/sub\u003e and F\u003csub\u003e2\u003c/sub\u003e seeds respectively as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The paternal seed colour expression in F\u003csub\u003e2\u003c/sub\u003e seeds might be attributed to the pollen mediated gene transfer/expression/interaction in quinoa. The evidences generated in current study were inconclusive. Therefore, in conclusion, further in-depth genetic, cytological and molecular analyses are required to better understand the seed colour inheritance in quinoa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Genetic improvement of quinoa with improved breeding methods and approaches\u003c/h2\u003e \u003cp\u003eOur study revealed the suitability of quinoa crossing approaches for diverse germplasms harbouring an array of floral diversity. In this study we have presented distinctive diagnostic traits that can help in improving efficiency of quinoa breeding, specifically differentiating characters of hermaphrodite flowers in some quinoa accessions. These distinctive traits enabled visual/ morphological identification of the true hybrids. In addition, we have observed the effectiveness of combined approach including both diagnostic and molecular markers in hybridity testing and genetic analysis that are important pre-requisites of a \u0026lsquo;breeding program\u0026rsquo;. The distinctive traits and floral arrangements of some accessions were so effective, that they facilitated \u0026ldquo;deliberate natural out-crossing\u0026rdquo; in quinoa, for example the black seed colour of accession, \u0026ldquo;D12377\u0026rdquo; or purple panicle colour of \u0026ldquo;Co 407\u0026rdquo;. Our findings suggest that large-scale identification of distinctive diagnostic characteristics in quinoa is urgently required.\u003c/p\u003e \u003cp\u003eA feasible quinoa breeding scheme has been presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. The success of the proposed breeding scheme underlies parental stock with distinctive diagnostic characteristics, for example, black seed, purple panicle, coloured hermaphrodite flowers etc. Secondly, the cross ability due to floral size and shape of parental lines is a critical parameter which significantly affects the natural crossing. The parental stock lines with distinctive diagnostic characteristics could be planned in a specific arrangement to facilitate natural crossing followed by selection of putative F1s based on key adaptive traits such as vigor, color, grain yield or earliness. Thereafter, generation advance can be made by breeding selections for preferred traits in every generation till sufficient homozygosity is attained. These fixed lines can then be subjected to multilocation yield stability trails as part of varietal pipeline. During this process, fixed genotypes with unique diagnostic characters can be identified and added to the parental stock for the next cycle of breeding advancements. Similar breeding schemes can be formulated based on identified distinctive diagnostic characteristics and pursued for breeding suitable varieties. These efforts are crucial for the global expansion of quinoa, particularly in newer environments of Asia and Africas.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe complex floral morphology of quinoa is the most important bottleneck in crossing and thereby its genetic improvement, which ultimately renders limited varietal diversity for varying crop ecologies in different parts of the world. The study presents the importance of the characterization of germplasm resources for the identification of the distinctive diagnostic traits that can be further utilized in overcoming crossing barriers. The study also validates the importance of \u0026lsquo;indel\u0026rsquo; markers in quinoa breeding, particularly the marker-assisted hybridity testing. In this research, an interesting pattern was observed related to the black seed color of quinoa, which requires further in-depth analysis to better understand the genetics behind it. In a nutshell, quinoa improvement for newer environments can be followed in a sequential manner starting from crossing block definition, distinctive diagnostic trait identification for overcoming crossing barriers to marker-assisted hybridity testing, and breeding selection for ecology-specific desired traits.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors hereby duly acknowledge funding support provided by the International Center for Biosaline Agriculture, UAE. Authors are also thankful to all intellectual and non-technical inputs provided by researchers at ICBA and other institutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePV, RKS and HR designed experiments; PV and SVJ were responsible for crossing, PV, LM, HR and SA performed hybridity testing; SNR and MGK were responsible for data gathering; SS, PV, GPM and RKS interpreted the results; PV drafted the manuscript. \u0026nbsp;All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/Additional Material. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was conducted by institutional research fund of the International Center for Biosaline Agriculture, UAE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJacobsen SE (1997) Adaptation of quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e) to Northern European agriculture: studies on developmental pattern. Euphytica 96:41\u0026ndash;48\u003c/li\u003e\n \u003cli\u003eJacobsen SE (2003) The worldwide potential of quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e Willd.). Food Rev Int 19:167\u0026ndash;177\u003c/li\u003e\n \u003cli\u003eJacobsen SE (2015) Adaptation and scope for quinoa in northern latitudes of Europe. In: FAO, CIRAD (eds) State of the art report on quinoa around the world in 2013. FAO, Rome, pp 436\u0026ndash;446\u003c/li\u003e\n \u003cli\u003eAmin QA, Wani TA, Nahvi AI (2022) Quinoa is a gluten-free, high-nutrient and underutilized crop with a lot of processing possibilities. Agric Food E-Newsl 2:3\u003c/li\u003e\n \u003cli\u003eAnderson JA, Churchill GA, Autrique JE, Tanksley SD, Sorrells ME (1993) Optimizing parental selection for genetic linkage maps. Genome 36:181\u0026ndash;186\u003c/li\u003e\n \u003cli\u003eBazile D, Jacobsen SE, Verniau A (2016) The global expansion of quinoa: trends and limits. Front Plant Sci 7:622. https://doi.org/10.3389/fpls.2016.00622\u003c/li\u003e\n \u003cli\u003eCollard BCY, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86\u0026ndash;93\u003c/li\u003e\n \u003cli\u003eCota-Sanchez JH, Remarchuk K, Ubayasena K (2006) Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue. Plant Mol Biol Rep 24:161\u0026ndash;167\u003c/li\u003e\n \u003cli\u003eEmrani N, Hasler M, Patiranage DSR, Maldonado N, Rey E, Jung C (2020) An efficient method to produce segregating populations in quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e). Plant Breed 139:1190\u0026ndash;1200. https://doi.org/10.1111/pbr.12858\u003c/li\u003e\n \u003cli\u003eFleming JE, Galwey NW (1995) Quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e). In: Williams JT (ed) Cereals and pseudocereals. Chapman and Hall, London, Underutilized Crops Series, vol 2, pp 3\u0026ndash;83\u003c/li\u003e\n \u003cli\u003eJames LEA (2009) Quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e Willd.): composition, chemistry, nutritional, and functional properties. Adv Food Nutr Res 58:1\u0026ndash;31\u003c/li\u003e\n \u003cli\u003eKoziol MJ (1991) Afrosimetric estimation of threshold saponin concentration for bitterness in quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e Willd.). J Sci Food Agric 54:211\u0026ndash;219\u003c/li\u003e\n \u003cli\u003eMurphy KM, Bazile D, Kellogg J, Rahmanian M (2016) Development of a worldwide consortium on evolutionary participatory breeding in quinoa. Front Plant Sci 7:608. https://doi.org/10.3389/fpls.2016.00608\u003c/li\u003e\n \u003cli\u003ePatirange DS, Rey E, Emrani N, Wellman G, Schmid K, Schm\u0026ouml;ckel SM et al (2022) Genome-wide association study in the pseudocereal quinoa reveals selection pattern typical for crops with a short breeding history. Elife 11:e66873. https://doi.org/10.7554/eLife.66873\u003c/li\u003e\n \u003cli\u003ePeterson A, Jacobsen SE, Bonifacio A, Murphy K (2015) A crossing method for quinoa. Sustainability 7:3230\u0026ndash;3243. https://doi.org/10.3390/su7033230\u003c/li\u003e\n \u003cli\u003eRahman H, Vikram P, Hu Y, Asthana S, Tanaji A, Suryanarayanan P et al (2024) Mining genomic regions associated with agronomic and biochemical traits in quinoa through GWAS. Sci Rep 14:19205. https://doi.org/10.1038/s41598-024-69992-2\u003c/li\u003e\n \u003cli\u003eSagar P, Chatterjee S, Joshi DC, Tiwari JK (2025) Standardization of different techniques for the rapid production of F1 hybrids in \u003cem\u003eChenopodium quinoa\u003c/em\u003e. Euphytica 221:123. https://doi.org/10.1007/s10681-024-03456-7\u003c/li\u003e\n \u003cli\u003eSha X (2013) Rice artificial hybridization for genetic analysis. Methods Mol Biol 956:1\u0026ndash;12\u003c/li\u003e\n \u003cli\u003eSimmonds N (1971) The breeding system of \u003cem\u003eChenopodium quinoa\u003c/em\u003e I. Male sterility. Heredity 27:73\u0026ndash;82\u003c/li\u003e\n \u003cli\u003eSingh D (2019) Quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e Willd.). Scientific Publishers, Jodhpur\u003c/li\u003e\n \u003cli\u003eTabatabaei I, Alseekh S, Shahid M, Leniak E, Wagner M, Mahmoudi H et al (2022) The diversity of quinoa morphological traits and seed metabolic composition. Sci Data 9:323. https://doi.org/10.1038/s41597-022-01435-8\u003c/li\u003e\n \u003cli\u003eWard S, Johnson D (1994) A recessive gene determining male sterility in quinoa. J Hered 85:231\u0026ndash;233\u003c/li\u003e\n \u003cli\u003eZhang T, Gu M, Liu Y, Lv Y, Zhou L et al (2017) Development of novel InDel markers and genetic diversity in \u003cem\u003eChenopodium quinoa\u003c/em\u003e through whole-genome re-sequencing. BMC Genomics 18:685. https://doi.org/10.1186/s12864-017-4003-2\u003c/li\u003e\n \u003cli\u003eZurita-Silva A, Fuentes F, Zamora P, Jacobsen SE, Schwember AR (2014) Breeding quinoa (\u003cem\u003eChenopodium quinoa\u003c/em\u003e Willd.): potential and perspectives. Mol Breed 34:13\u0026ndash;30\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Quinoa, Pseudocereals, breeding, marker-assisted breeding, genetic enhancement","lastPublishedDoi":"10.21203/rs.3.rs-9391447/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9391447/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eQuinoa represents a valuable gift from the \"New World\" to the \"Old World.\" Although some breeding programs exist worldwide, they have seen limited growth and impact on its genetic improvement. Large-scale hybridization, hindered by the crop's complex floral morphology, remains the primary bottleneck in quinoa breeding. This study compared manual and natural crossing schemes, incorporating hybridity testing with morphological and molecular markers. The results indicate that a facilitated open-pollinated strategy combined with hand emasculation and hybridity confirmation using morphological or indel markers offers a feasible approach to establishing a quinoa breeding pipeline. We also concluded that characterising panicles by the arrangement of hermaphrodite and pistillate flowers could be a game-changer for improving breeding efficiency. Additionally, interspecific crosses (Chenopodium quinoa \u0026times; Chenopodium giganteum) achieved efficiencies of 5\u0026ndash;26%. Both inter- and intraspecific crosses can feasibly generate variation for genetic improvement in quinoa. Breeding quinoa for Asian environments should follow a step-wise strategy: (1) characterize flowering in crossing blocks, (2) apply natural crossing or hand emasculation, (3) confirm hybridity using morphological or molecular markers, (4) pursue genetic enhancement, and (5) select for traits of interest.\u003c/p\u003e","manuscriptTitle":"Strategic Breeding Approaches: Harnessing Molecular and Phenotypic Markers to Overcome Quinoa's Flower Morphology Bottlenecks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 13:10:35","doi":"10.21203/rs.3.rs-9391447/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-15T07:03:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291864793178748740570314227134128875820","date":"2026-05-14T14:46:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T15:54:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158160565066767274725470498357920805712","date":"2026-04-20T08:22:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299431298562346765159166319377461102286","date":"2026-04-18T17:19:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T16:36:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-17T11:46:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T07:22:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T07:22:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-12T03:39:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"40009153-2f23-4890-9fa9-05bf85c9ee1e","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-15T07:03:04+00:00","index":46,"fulltext":""},{"type":"reviewerAgreed","content":"291864793178748740570314227134128875820","date":"2026-05-14T14:46:32+00:00","index":45,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67090400,"name":"Biological sciences/Biological techniques"},{"id":67090404,"name":"Biological sciences/Biotechnology"},{"id":67090405,"name":"Biological sciences/Genetics"},{"id":67090406,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-04-27T13:10:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 13:10:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9391447","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9391447","identity":"rs-9391447","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}