Introduction
Discovery of opaque and other related mutants in maize is considered as a land mark achievement of 20th century. However, developing the quality protein maize (QPM) which is the cultivable and practical version of opaque mutant maize was a daunting task which was produced through eventual discovery and deployment of endosperm modifiers by Villegas (Reference Villegas1964). Despite this achievement and co-popularity of single cross hybrids and subsequent release of various QPM hybrids it was observed that due to trait compensation QPM hybrids have never been able to reach within the range of normal maize hybrids in terms of grain yield.
Babu et al. (Reference Babu, Nair, Kumar, Venkatesh, Sekhar and Singh2005) for the first time in India devised a plan of transferring the opaque gene rapidly through marker assisted backcrossing in the normal inbred parents of established hybrids. So that, these backcross converted lines can be used to recreate the QPM version of elite hybrids. These attempts led to the creation of landmark hybrids such as, Vivek QPM-9, HQPM-1, 2, 3 and 4 etc. However, the backcrossing programme involves confirmation of genetic foreground as well as recovery of genetic background of recipient lines/parents. This led to the combination of SSR marker based pre-selection of progenies at vegetative stage and conventional backcross at flowering stage so that crosses can be only made in the plants after verification of their genetic composition and one extra cycle of planting and progeny testing can be avoided. Hybrid versions created through this method possess yields within the range of their normal versions and have tryptophan content more than 0.075% (in sample).
In Jammu and Kashmir such an attempt is required considering the popularity of maize as a staple crop and its potential in the government sponsored Mid Day Meal Scheme for school children of poor families. The QPM doesn't have visible differences from normal maize and the QPM trait is also qualitative in marketing sense. Therefore, farmer adoption is quite an upward slope as of now. Other than thrust on use of QPM in mid-day meal scheme in India, there is no current possibility of Government of India drafting a policy for popularizing the QPM in domestic agri-industry. The poultry, milk and meat industry is about 12 million USD big in Jammu and Kashmir (ASH & FD, JK, 2022). Maize is consumed mainly as livestock feed and fodder which definitely creates QPM demand because poultry farmers and enterprises have to provide the limiting amino acids through supplementation. The bio-fortification of maize cultivars has proved benefits on human health (Tessema et al., Reference Tessema, Gunaratna, Donato, Cohen, Connell, Belayneh, Brouwer, Belachew and Groote2016)
Therefore, an attempt was made at SKUAST-Kashmir to convert the parental lines of Shalimar maize hybrid-5 (9 ton/ha grain yield) into QPM versions retaining original genetic background using marker assisted backcross shuttle breeding. However, an initial cross with a suitable synchronic donor and subsequent confirmation of heterozygosity is required through use of o2 gene based SSR markers. Three such PCR based markers viz. phi 057, phi 112 and umc 1066 are known to show polymorphism as well as a Mendelian segregation between QPM and normal maize via gel electrophoresis. The polymorphic & co-dominant of these can find heterozygote which is the only purpose of MAS in foreground selection. There are intricacies of finding recurrent parent genome in backcross F1s using molecular markers before we can self-pollinate the plants for recessive allele fixation. Genome wide SSR markers can be used to identify recurrent parent genome similarity in the converted O2o2 (heterozygote) after at least two backcrosses (Hossain et al., Reference Hossain, Muthusamy, Pandey, Vishwakarma, Baveja, Zunjare, Thirunavukkarasu, Saha, Manjaiah, Prasanna and Gupta2018). The preference of SSR markers is understood due to their low cost. Hossain et al. (Reference Hossain, Jaiswal, Muthusamy, Zunjare, Mishra, Chand, Bhatt, Bhat, Das, Chauhan and Gupta2023) explained in detail the rationale behind use of SSR markers instead of more dispersed in genome SNPS (SSR~0.25$ per sample; KASP~4$ per sample; GBS~50$ per sample; WGS~400$–1000$ per sample). Although, SSR genome coverage is low but their efficiency in background selection is very well received (Kumar et al., Reference Kumar, Rai, Singh, Kumar, Ahlawat, Saini, Shukla, Bedi and Singh2022; Rajarathinam et al., Reference Rajarathinam, Palanisamy, Ramakrishnan, Narayana and Alagirisamy2023). Use of MAS before elucidation of phenotypic DUS based similarity with recurrent parents is recommended because the frequency of parental type phenotype increases after SSR marker based rouging thus reducing the physical labour of field based phenotypic background selection to a considerable extent.
Conversion of SMH-5 into a QPM version is important because this hybrid has a yield potential of 9–10 t/ha. If the converted hybrid can have a yield at par to SMH5 with better protein quality, a major bottleneck of QPM cultivation which is lower grain yield can be avoided. Also a rapid backcross breeding strategy needs to be established specifically for Kashmir valley where only one crop of maize is taken in a year and breeding for new varieties takes several years.
Materials and methods
Breeding programme
The programme was carried out using shuttle breeding method from 2019–2022 at Faculty of Agriculture Wadura SKUAST-Kashmir J&K, India (34°35′22″N, 74°40′03″E), Winter Nursery Centre, IIMR, Rajendra Nagar Hyderabad, Telangana (17°32′22″N and 78°40′06″E). India and Agriculture Research Station Peddapuram, Andhra Pradesh, India (17°07′N and 82°14′E) in a biannual crossing programme. IML-187 (ICAR-IIMR Ludhiana) and BML-6 (PJTSAU Hyderabad), originally the parents of Shalimar maize hybrid-5 were utilized as recurrent parents (O2O2) whereas; DQL-2029-1 and DQL-779-2-9 (IIMR Ludhiana) were used as donor parents (o2o2). The F1s were generated during rabi (winter) season (December 2019–April 2020) at WNC Hyderabad. These F1s were raised at Peddapuram Andhra Pradesh during kharif season (July–November 2020) and true hybrids were backcrossed to respective recurrent parents. The backcross generations BC1F1, BC2F1 and BC2F2 were planted respectively at winter nursery Hyderabad (rabi December 2020–April 2021), Faculty of Agriculture Wadura, SKUAST-Kashmir (kharif, May–November 2021) and winter nursery (rabi December 2021–April 2022). Foreground selection was performed at BC1F1 and BC2F1 stage after using SSR marker selected on the basis of parental polymorphism. Chi square test was performed to test segregation ratio of marker positive and negative plants in BC1F1 generation. Background selection was done in opaque 2 recessive (o2o2) homozygote BC2F2 generation using polymorphic SSR markers identified on the basis of polymorphism among parents. Phenotypic selection of converted progenies was conducted using DUS descriptors (online Supplementary Table S1) (PPV & FRA of INDIA, 2007).
DNA isolation and PCR
The marker assisted backcross scheme is presented in Fig. 1. DNA isolation was done using DNeasy™ Plant Mini Kit (EN) (QIAGEN Inc. Germany). PCR reaction was carried out for 40 background SSR markers and 3 opaque 2 linked SSRs (online Supplementary Table S2). The band visualization and marker based selection was done using standard gel electrophoresis. The amplified products (3 μl) were resolved on a 3.5% agarose gel prepared by dissolving 3.5 g of agarose gel in 1X TAE buffer of 100 ml and boiling it for 2 min in microwave oven and 2 μl EtBr was added at 60°C temperature. The gel was casted in the gel tray of electrophorosis unit after solidifying kept in the gel tank filled with 1X TAE. The documentation of SSR profile was done using 100 bp ladder in the first tank while rest of PCR products in rest of the lanes and each entry was recorded after running at 80 V for 2 h. After the gel run was completed, it was placed on the platform of gel documentation system and under Chemi Doc Imaging System (ChemiDoc™ Imaging System #12003153; Bio-Rad Laboratories, Inc), the photograph was taken. Each gel was labelled properly and date record was kept.
Figure 1. Breeding programme for marker assisted backcross breeding to enhance lysine and tryptophan content of Shalimar maize hybrid-5.
Nutritional analysis
Tryptophan % (in sample) was estimated using the Acquity UPLC H-class plus system (WATERS™ 159 Lukens Drive New Castle, DE 19720) at biochemistry laboratory, CAR- IIMR Ludhiana (INDIA). Aliquot was prepared using Quecher's method (Sarika et al., Reference Sarika, Hossain, Muthusamy, Zunjare, Baveja, Goswami, Bhat, Saha and Gupta2018) in BC2F3 seed. Lysine (in protein) was calculated by multiplying tryptophan content in sample (Pixley and Bjarnason, Reference Pixley and Bjarnason1993). The resultant value was an approximate estimation of lysine per cent in protein. The selection of plants for tryptophan and lysine was done after one factor analysis using Completely Randomized Design (CRD) (Cochran and Cox, Reference Cochran and Cox1959). A minimum selection criterion of 0.075% tryptophan and a quality index of 0.8 were used. Quality Index is the ratio of tryptophan content to the total protein content (%) in the sample.
Selection of vitrous kernels
Grain opaqueness study was done at BC2F3 seed at Faculty of Agriculture Wadura. The selected plants were subjected to kernel modification analysis using light box method. Screening of kernels in light box categorized four degrees of opaqueness viz. <25, 25–50, 50–75 and 75–100% Krishna et al. (Reference Krishna, Reddy and Satyanarayana2017).
Results and discussion
Parental polymorphism for molecular and morphological markers
In the present study, it was observed that phi 112 marker exhibited dominant behaviour between QPM and non-QPM lines. The phi 112 allele was only observed in normal maize as it is O2O2 and was absent in QPM lines. Therefore, this marker cannot be used in a conversion programme but it is usable in determining the purity of QPM varieties. The markers phi057 and umc1066 exhibited co dominant polymorphism between IML-187, BML6 and DQL-779-2-9, DQL-2029-1. The marker phi057 amplified at 160 bp in IML-187(P1) and 160 bp in BML-6 (P2) whereas an allele size of 170 bp was observed in DQL-2029-1 (P3) and DQL-779-2-9 (P4) (online Supplementary Table S3 and Fig. S1) and the occurrence of polymorphic alleles at o2 locus clearly distinguishes these lines from one another on the basis of opaque gene. Similar results have been reported earlier by Bantte and Prasanna (Reference Bantte and Prasanna2003) who noted clear and discrete polymorphism at o2 locus. Therefore the use of phi 057 marker in identifying the desired o2 individuals in the F1 and subsequent backcross progenies is advisable (Shetty et al. (Reference Shetty, Sagare, Surender and Reddy2020)). The polymorphism of 40 background SSR markers for each parent is depicted in the online Supplementary Table S3 and Figs. 2–10. Out of these 25 markers were found to be polymorphic. Kostadinovic et al. (Reference Kostadinovic, Nikolic, Ristic, Bozinovic, Melnik, Micic and Vancetovic2019) used 30 SSR markers for background selection during introgression of favourable alleles in 12 maize inbred lines to determine genetic similarity and percentage of recurrent parent genome. They were able to obtain genetic similarity values ranging from 79 to 99. 48% of progenies had above theoretical values and were self-pollinated for allele fixation. Parental polymorphism on the basis of DUS descriptors/morphological markers was also determined prior to the inception of breeding programme. Phenotypic selection coupled with marker assisted selection results in maximum gain in terms of recurrent parent similarity. The phenotypic trait expression of the parents is described in the online Supplementary Table S1.
Selection of plants from BC1F1 to generate BC2F1
The F1 hybrids were crossed with recurrent parents to generate BC1F1 (A) and BC1F1 (B). And seeds were sown to raise BC1F1 plant populations. Initially 50 plant populations from each of the BC1F1 (A) and BC1F1 (B) seeds were obtained, however seeds of 25 and 28 plants were retained from BC1F1(A) and BC1F1 (B) respectively. These BC1F1s were sown in 4 m row pattern to raise approximately 200 plants each, along with two rows of recurrent parents. However, only 96 plants from BC1F1 (A) and 100 plants from BC1F1 (B) were genotyped to determine the heterozygosity using phi 057 SSR primer (online Supplemantary Figs. 11 and 12). Significant χ 2 values were observed in both the crosses indicating Mendelian test cross ratio of 1:1. The number of heterozygote plants is mentioned in online Supplementary Table S4. However due to poor seed set and phenotypic selection pressure, only 20 and 23 plants from BC1F1(A) and BC1F1 (B) respectively were backcrossed to generate BC2F1 seeds. Magulama and Sales (Reference Magulama and Sales2009) noted polymorphism at o2 locus with phi057 and umc1066 however they applied only phi057 in marker assisted selection for the development of BC populations. Because of the reliability and discrete polymorphism, phi057 SSR marker has also been used in marker assisted backcrossing studies earlier by Manna et al. (Reference Manna, Okello, Imanyhowa, Pixley and Edema2005) and Danson et al. (Reference Danson, Mbogori, Kimani, Lagat, Kuria and Diallo2006). The results of Jompuk et al. (Reference Jompuk, Cheuchart, Jompuk and Apisitwanich2011) were also on the same line as observed in the present investigation since genotyping of o2 locus using phi057 exhibited allele size of 160 and 170 bp in o2 and non-opaque-2 maize lines, respectively. However, Gupta et al. (Reference Gupta, Raman, Agrawal, Mahajan, Hossain and Thirunavukkarasu2013) used umc1066 successfully in foreground selection for o2 allele. The allele size they observed was 460 bp in normal maize line whereas allele of 480 bp was found in QPM donor line using marker umc1066.
Selection of plants from BC2F1 to generate BC2F2
Total of 200 seeds each from selected BC2F1 (A) from BC2F1 (B) were sown separately. One row of recurrent parent was sown after every two rows of BC2F1 plants to facilitate selection of plants similar to recurrent parents. The plants left in the field after pre-flowering screening were tagged and leaf samples from individual plant were collected and genotyped for o2 locus using phi-057 SSR marker (online Supplementary Figs. 13 and 14). The genotyping at o2 locus helped in identifying plants heterozygous for o2 locus since population in BC2F1 consisted of both heterozygous and homozygous plants in 1:1 ratio. Significant χ 2 values were observed in both the crosses indicating Mendelian test cross ratio of 1:1. 36 and 44 plants were observed to be heterozygous at o2 locus from BC2F1 (A) and BC2F1 (B) respectively (online Supplementary Table S5). The heterozygous plants were also genotyped for recurrent parent genome using 25 polymorphic SSR markers. At flowering, plants were again screened for phenotypic traits mentioned in online Supplementary Table S1 (as in the previous generation) similar to recurrent parents and off types were rogued out. The final number of plants selfed (18 and 16) to produce BC2F2 (A and B) for each progeny after phenotyping and genotyping are presented in online Supplementary Table S5. As stated earlier by Babu et al. (Reference Babu, Nair, Kumar, Venkatesh, Sekhar and Singh2005), identification of heterozygotes in the seedling stage prior to pollination aids in the rejection of non-target BC progenies, resulting in substantial saving of labour and material resources. Magulama and Sales (Reference Magulama and Sales2009) developed the two BC populations to employ marker assisted selection for o2 gene. Gupta et al. (Reference Gupta, Raman, Agrawal, Mahajan, Hossain and Thirunavukkarasu2013) identified heterozygous (O2o2) progenies that occurred with 50% frequency in a given backcross population.
Approximate percentage genome similarity to recurrent parents in selected lines using polymorphic background SSR markers
The 18 and 16 plants of BC2F1 (A) and (B) were selfed after foreground and background selection in previous generation. Their seeds (BC2F2) were harvested and sown in 4 m row system. 100 BC2F2 plants were raised each from both progenies were first studied for marker segregation at o2 locus (online Supplementary Figs. 15 and 16) to performforeground selection for recessive homozygotes. The marker segregation ratio at this locus was close to Mendelian monohybrid genotypic ratio of 1:2:1 (using X 2 test). The selected recessive homozygotes were subjected to study recurrent parent genome similarity which is called as background selection. Background genome recovery among the BC2F2 progenies of both the crossing schemes was performed using 25 polymorphic SSR markers. In the present study we selected those entry numbers, or plants which had recurrent parent similarity for at least 20 background markers. The entry number selected were IML-187 × DQL-2029-1/BC2F2-1,2,3,36,15,6,7,8,31,19,11,29,27,14,5,16,17 and 23 and for BC2F2 (B) were BML-6 × DQL-779-2-9/BC2F2-1, 3,7,44,31,28,27,18,13,10,23,8,20,4,14 and 22 (Table 1). Only these entries were selfed to generate BC2F3 seeds. Including a selection step on non-carrier chromosomes is more effective in later backcross generations (BC3 or BC4) than in early generations. Although this may be counter intuitive, it is based on the facts that in early generations, few recombination have occurred to break up introgressed segments from the donor thus, few markers are needed, and that most of the donor material will be removed in subsequent backcrosses. Therefore a smaller number of background markers can suffice in early backcross generations such as BC2F1 and BC2F2. Also, the 15 SSR markers being non polymorphic mean that genome of recurrent and donor parents is similar at these loci. Thus selection for the areas related to non-polymorphic markers is already been done by nature. Therefore the overall genome selected will be more than what noticed on the basis of 25 polymorphic markers. Our selection procedure was assisted by phenotypic selection as well. Those traits have also assisted in selection as phenotypic markers. In later generations, the donor regions will be smaller, and hence more markers will be needed. Selection against donor alleles in the region of the target i.e. reducing the linkage block surrounding the target has been discussed by numerous authors (Frisch et al., Reference Frisch, Bohn and Melchinger1999; Reyes-Valdés, Reference Reyes-Valdés2000; Frisch and Melchinger, Reference Frisch and Melchinger2001; Hospital, Reference Hospital2001). The goal is to reduce the size of the donor DNA segment surrounding the target gene to as small as possible in the backcrossing programme. Marker assisted background analysis of the BC/recombinant progenies is useful in determining the relative contribution of donor parent. Availability of robust anchored marker maps in maize renders application of marker aided background selection an easy and attractive proposition. However, the cost of employing background selection in each BC generation could be prohibitive to many public sector breeding programmes. The earlier simulation studies of Frisch et al. (Reference Frisch, Bohn and Melchinger1999) have indicated that application of background selection in one later generation along with foreground selection in each BC generations could be efficient and cost-effective. Chandran et al. (Reference Chandran, Pukalenthy, Adhimoolam, Manickam, Sampathrajan, Chocklingam, Eswaran, Arunachalam, Joikumar, Rajasekaran, Muthusamy, Hossain and Natesan2019) utilized genome wide SSR markers to recover background genome in BC2F2 population. Hospital et al. (Reference Hospital, Chevalet and Mulsant1992) stated that care needs to be taken in a practical MAS programme to avoid sampling error and exaggerated estimates of recurrent parent genome associated with smaller number of marker data points.
Table 1. Percentage genome similarity of BC2F2 (A) with IML-187 and BC2F2 (B) with BML-6 based on 25 polymorphic SSRs
Phenotyping of BC2F3 seeds for endosperm modification using light box
The BC2F3 seeds harvested from selfing of BC2F2 plants in previous generation were kept separate as entries and were screened for vitreous endosperm. Screening of kernels in light box categorized four degrees of opaqueness viz. <25, 25–50, 50–75 and 75–100% Krishna et al. (Reference Krishna, Reddy and Satyanarayana2017) (online Supplementary Fig. S12). The number of plants used for screening in light box and finally selected is presented in online Supplementary Table S6. The screening of the seeds was done using light box having fluorescent light with endospermic region upside down in the dark room. The high lysine and tryptophan in QPM is due to the presence of opaque 2 gene in homozygous recessive state. The endosperm of opaque 2 maize has variable degree of opaqueness and this trait is governed by endosperm modifiers. The inheritance of endosperm modifiers is polygenic in nature and there is no reliable molecular marker to accurately genotype these genes. These modifier genes do not have effect of their own but have an interaction effect on opaque mutants and thus modify kernel opaqueness (Vasal et al., Reference Vasal, Villegas, Bjarnason, Gelaw, Goertz, Pollmer and Phillips1980). Lopes and Larkins (Reference Lopes and Larkins1995) showed that kernel modification in QPM is governed by more than one minor gene. As a result, each kernel has different degree of modification due to action of many genes with small effect. Of the differentially modified kernels, those exhibited less than 25% opaqueness were selected from each ear whereas kernels over 25–50% and more than 50% opaqueness were rejected due to soft nature and dull appearance of kernels (Vasal et al., Reference Vasal, Srinivasan, Pandey, González, Crossa and Beck1993). Thus the screening of seeds using a light box is a high throughput and easy way to differentiate kernels on the basis of extent of opaqueness. This helps to select QPM kernels with endosperm similar to normal parents.
Determination of protein, tryptophan and lysine content in the BC2F3 seeds
The seeds from the selfed BC2F2 population of the selected lines were harvested as BC2F3 seeds after undergoing endosperm modification screening were analysed individually along with their parental lines for estimation of protein, tryptophan and lysine content. The estimated per cent (%) value of nitrogen obtained using Rapid N-cube Analyzer instrument was used to determine the protein content in per cent (Elementar Analysensysteme GmbH, web: www.elementar.de, D 63452 Hanau, Germany). The estimated per cent protein, tryptophan and lysine content values of the converted lines developed from the IML-187 × DQL-2029-1 BC2F3 and BML-6 × DQl-779-2-9 BC2F3 are given in the online Supplementary Table S7. The results indicated the lowest tryptophan value of 0.047% in IML-187 × DQL-2029-1 BC2F3:12 line while the maximum value of 0.117% was found in BML-6 × DQl-779-2-9:12. The four parents namely IML-187, BML-6, DQL-2029-1, and DQL-779-2-9 used in conversion programme had 0.045, 0.042, 0.074 and 0.090% tryptophan, respectively (online Supplementary Table S7). Thus, wide variation for tryptophan content was observed in recombinant BC2F3 lines. The criteria used in QPM breeding for selection or rejection of breeding lines include tryptophan content which should be 0.075% or more on whole grain sample basis and quality index (QI) of 0.8% or more (Vivek et al., Reference Vivek, Krivanek, Palacios-Rojas, Twumasi-Afriyie and Diallo2008). The quality index is the tryptophan-to-protein ratio in the sample, expressed as a percentage. Values lower than this indicate a non QPM cultivar. Using this yardstick, eight lines namely {IML-187 × DQL-2029-1- BC2F3:06}, {IML-187 × DQL-2029-1- BC2F3:07}, {IML-187 × DQL-2029-1- BC2F3: 23} and {BML-6 × DQL-779-2-9: 02}, {BML-6 × DQL-779-2-9: 04}, {BML-6 × DQL-779-2-9: 09}, {BML-6 × DQL-779-2-9: 20} and {BML-6 × DQL-779-2-9: 13} were identified to have tryptophan higher than 0.075%. Line {IML-187 × DQL-2029-1- BC2F3:23} was selected due to better lysine and overall protein content despite having 0.074% tryptophan content. Studies by Gupta et al. (Reference Gupta, Raman, Agrawal, Mahajan, Hossain and Thirunavukkarasu2013) pointed out that the o2 allele when present in homozygous condition reduces the production of zeins, particularly the α-zeins, through which it increases the level of lysine and tryptophan. The presence of o2 modifiers will contribute to the recovery of hard endosperm. This supposition is because of experimentally and statistically well proven and well established relationship between the tryptophan and lysine. Lysine and tryptophan levels are highly correlated and as such an assay for either amino acid can be used for analysing protein quality, although in practice the latter is most often chosen due to lower laboratory costs (Hernandez and Bates, Reference Hernandez and Bates1969; Villegas et al., Reference Villegas, Vasal, Bjarnason and Mertz1992). Lysine and tryptophan concentration in maize kernels of agronomically advanced QPM lines are highly correlated. A 4:1 ratio of lysine to tryptophan has been reported in normal and QPM (Bjarnason and Vasal, Reference Bjarnason, Vasal and Janick1992; Vivek et al., Reference Vivek, Krivanek, Palacios-Rojas, Twumasi-Afriyie and Diallo2008).
Crossing between converted BC2F3 progenies to produce QPM version of SMH-5
The selected BC2F3 entries were sown in kharif 2022 and crosses were made between converted progenies of IML-187 and BML-6. The crosses were made in a Line × Tester pattern using three selected entries from IML-187 progeny as female tester parents and five selected entries of that of BML-6 progeny as male lines. Crosses were evaluated in kharif- 2023 at Dryland Agricultural Research Station, Rangreth SKUAST-Kashmir (33°99′04″N and 74°80′65″E). The crosses and their performance for protein, tryptophan and yield/ha is presented in online Supplementary Table S8.
The varietal development programmes take several years. The marker assisted backcross breeding with two generations per year is a useful tool in transferring single or a few traits to the adapted elite lines. This procedure reduces the time taken in the development of improved versions of existing varieties/inbred lines by half. Thus, hastening the hybrid development programmes. In the present study, three improved versions of IML-187 and five versions of BML-6 were developed with a genome recovery of 90% based on 25 polymorphic SSR markers. It should be noted that 15 additional loci were similar to recurrent types due to monomorphism. Finally, 15 improved versions of SMH-5 were developed in the present study as presented in online Supplementary Table S8. Hybrids, {IML-187: BC2F3:6 × BML-6: BC2F3:2},{IML-187: BC2F3:6 × BML-6: BC2F3:13}, {IML-187: BC2F3:6 × BML-6: BC2F3:20}, {IML-187: BC2F3:7 × BML-6: BC2F3:9}, {IML-187: BC2F3:23 × BML-6: BC2F3:2}, {IML-187: BC2F3:23 × BML-6: BC2F3:4} are being considered for release in the early maturity group in northern hill zone of India, indicating Marker assisted shuttle backcross breeding is a reliable, rapid and convenient tool in development of biofortified elite adapted crop varieties.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262124000194.
Data
The data regarding sequences of SSR markers used in the present study was downloaded from Maize Genome Database (https://www.mgdb.org).
Acknowledgements
We thank NSP lab SKUAST-K, Shalimar for providing the field and lab facility. ICAR-IIMR, Ludhiana, is acknowledged for providing the off-season nursery at WNC, Hyderabad and lab for bio-molecular analysis. ARS Peddapuram for providing resources for growing of field trials. We also thank Maize research station, PJTSAU Rajendra Nagar Hyderabad for providing lab facilities.
