Introduction
Sorghum [Sorghum bicolor (L.) Moench] is the fifth most important cereal crop cultivated in the arid and semi-arid tropics of the world for food, feed and fodder and in the recent past, has been promoted as a bio-energy crop. Sorghum has one of the largest germplasm collections, comprising more than 42,000 accessions (Dahlberg et al., Reference Dahlberg, Zhag, Hart and Mullet2002; Huang, Reference Huang2004). Miniaturization of germplasm collections with the maximum representation of genetic diversity in the form of mini-core (~1% of the entire collection) is an effective method to enrich and augment crop breeding programmes (Upadhyaya et al., Reference Upadhyaya, Yadav, Dronavalli, Gowda and Singh2010). In sorghum, 242 mini-core collections have been reported to constitute from a set of 2247 core originating from 58 countries (Upadhyaya et al., Reference Upadhyaya, Pundir, Dwivedi, Gowda, Reddy and Singh2009).
The rich diversity present among sorghum germplasm accessions is due to the presence of diverse races and geographical origins. Cultivated races include five basic races (bicolor, guinea, caudatum, kafir and durra) and ten intermediate races (Harlan and de Wet, Reference Harlan and de Wet1972). The races are also linked to specific growing environments (Smith and Frederiksen, Reference Smith and Frederiksen2000). Africa, being the centre of origin of the crop, harbours the richest diversity across the world (Doggett, Reference Doggett1970; de Wet and Harlan, Reference de Wet and Harlan1971; de Wet, Reference de Wet1977). Sorghum in India is cultivated during rainy and post-rainy seasons with cultivars specifically adapted to each season. Rainy season cultivars are predominantly caudatum, kafir and bicolor races, while post-rainy season cultivars are mainly the durra types. Heterosis is best exploited in rainy season cultivars, but not to a greater extent in post-rainy season cultivars, which is mainly due to the narrow genetic base of the post-rainy genotypes (Sajjanar et al., Reference Sajjanar, Biradar and Biradar2011). During the last decade, significant yield enhancement could not be achieved in both rainy and post-rainy sorghum groups, which is mainly attributed to the narrow genetic base of the breeding materials, and this has warranted an urgent need for genetic improvement by the utilization of germplasm from diverse sources. For genetic improvement, knowledge on the genetic diversity and genetic structure in the germplasm materials is very essential. Both morphological and molecular methods are the commonly used techniques for the assessment of genetic diversity. In sorghum, application of molecular markers for studying the genetic diversity and genetic structure has been carried out by different research groups (Dje et al., Reference Dje, Heuertz, Lefebvre and Vekemans2000; Ghebru et al., Reference Ghebru, Schmidt and Bennetzen2002; Agrama and Tuinstra, Reference Agrama and Tuinstra2003; Anas and Yoshida, Reference Yoshida2004; Muraya et al., Reference Muraya, Mutegi, Geiger, de Villiers, Sagnard, Kanyenji, Kiambi and Parzies2011; Rakshit et al., Reference Rakshit, Gomashe, Ganapathy, Elangovan, Ratnavathi, Seetharama and Patil2012; Ramu et al., Reference Ramu, Billot, Rami, Senthilvel, Upadhyaya, Ananda Reddy and Hash2013; Wang et al., Reference Wang, Jiao, Jiang, Yan, Su, Sun, Yan and Sun2013b; Lekgari and Dweikat, Reference Lekgari and Dweikat2014). Studies have been conducted to assess the genetic diversity of mini-core collection; however, few attempts have been made to compare its diversity with the breeding lines for its wide utilization in breeding programmes.
In view of the above importance, the present study aimed at assessing the extent of SSR diversity within the sorghum mini-core and elite Indian genotypes (rainy and post-rainy sorghum). The objectives of this study were (1) to assess the genetic diversity and genetic structure of the mini-core and the elite genotypes of rainy and post-rainy season adaptation of India, and (2) to compare the genetic diversity between the mini-core and elite genotypes and to identify diverse germplasm accessions for their use in breeding programmes.
Materials and methods
A total of 251 sorghum genotypes comprising 199 mini-core, 44 elite sorghum genotypes and eight elite germplasm were used in the present study. The mini-core for the study was obtained from the Gene Bank of the Indian Institute of Millets Research, Hyderabad, which in turn was provided by ICRISAT, Hyderabad, India. Race and the passport information of the mini-core collection available in the sorghum germplasm database of ICRISAT (http://www.icrisat.org/what-we-do/crops/sorghum/Project1/pfirst.asp) were used for the study. The sorghum mini-core included a total of 242 accessions, of which only 199 were included in our study. The remaining 43 accessions were eliminated from the present study as they were photo-sensitive/failed to flower during the cropping season and or had poor plant stand. Therefore, we complemented the mini-core set with a set of 44 elite and 8 germplasm lines that were already characterized for agronomic and grain quality traits. The 44 elite genotypes chosen represented the diversity present among the Indian breeding lines. Of these elite lines, 25 belonged to the rainy season adaptation and 19 belonged to the post-rainy season adaptation. The 25 rainy sorghum types included 4 varieties, 6 maintainer lines, 9 restorer lines and 6 advanced breeding lines. The 19 post-rainy sorghum lines included 6 varieties, 9 advanced breeding lines and 4 landraces (see online supplementary, Table S1).
DNA extraction and SSR analysis
Genomic DNA was extracted using the CTAB (cetyl trimethyl ammonium bromide) protocol according to the method described by Rakshit et al. (Reference Rakshit, Gomashe, Ganapathy, Elangovan, Ratnavathi, Seetharama and Patil2012). The quantity and quality of DNA was assessed by taking spectrophotometer readings at 260 and 280 nm, and stock DNA was diluted to make a working solution of 5 ng/μl for PCR analysis.
For SSR analysis, the SSR diversity kit representing sorghum linkage groups (http://sat.cirad.fr/sat/sorghum_SSR_kit/) was used for the study. Among the 48 markers used in this study, 39 reliable markers were used for analysis. The analysis was carried out at Genotyping Services Laboratory of ICRISAT, Patancheru, India. Capillary electrophoresis was carried out using the ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster city, CA, USA). The procedure has been described in detail by Ganapathy et al. (Reference Ganapathy, Gomashe, Rakshit, Prabhakar, Ambekar, Ghorade, Biradar, Saxena and Patil2012). The data generated were then analysed using GeneMapper software (Applied Biosystems, Foster city, CA, USA), and fragment size was scored in base pairs (bp) based on the relative migration of the internal size standard.
Data analysis
The allelic data obtained from GeneMapper software were used for estimating diversity parameters such as number of alleles (N), major allele frequency (A), observed heterozygosity (H o), expected heterozygosity/gene diversity (H e) and polymorphism information content (PIC) for each SSR locus. Gene diversity, as defined by Weir (Reference Weir1996), is the probability of two randomly chosen alleles being different from a population. PIC (Botstein et al., Reference Botstein, White, Skolnick and Davis1980) is the measure to calculate the discrimination power and informativeness of SSR markers.
The STRUCTURE program (http://pritch.bsd.uchicago.edu/structure.html) was used to assess the population structure among 251 accessions (Pritchard et al., Reference Pritchard, Stephens and Donnelly2000; Falush et al., Reference Falush, Stephens and Pritchard2007). The program applies a model-based clustering algorithm that implements the Markov chain Monte Carlo algorithm and a Bayesian framework. The optimum number of subpopulations (K) was identified after four independent runs for each value of K ranging from 2 to 10, with a burn-in of 50,000 iterations followed by 100,000 iterations. The algorithm identifies subgroups with distinctive allele frequencies, and places individuals into K clusters using its estimated membership probability (Q). For classifying the individuals into groups, a membership coefficient of above 0.8 was considered as a feasible cut-off membership value to assign individuals to a population with confidence. The results were post-processed with the ad hoc approach of Evanno et al. (Reference Evanno, Regnaut and Goudet2005) to estimate the number of genetic clusters using Structure Harvester (http://taylor0.biology.ucla.edu/structureHarvester/; Earl and vonHoldt, Reference Earl and vonHoldt2012). The height of the modal value generated from the structure harvester analysis was used to identify the number of subgroups detected by the structure analysis.
For calculating the diversity parameters, namely frequency-based genetic distance and population-specific F statistic, the mini-core collections were classified based on their race and geographical origin. Genotypes with substantial population size for races were used for F Statistic and Nei genetic distance analysis. The 44 elite Indian sorghum genotypes were classified into rainy and post-rainy sorghum groups. The frequency-based pairwise genetic distance was calculated as given by Nei (Reference Nei and Morton1973), while the population specific F statistic was calculated according to Weir and Hill (Reference Weir and Hill2002). All the above parameters were analysed using Powermarker ver. 3.25 (Liu and Muse, Reference Liu and Muse2005). Clustering of individual accessions based on race and geographical origin was carried out using the neighbour-joining method implemented in DARwin 5.0 software (Perrier and Jacquemoud-Collet, Reference Perrier and Jacquemoud-Collet2006).
Results
Genetic diversity estimates using SSR markers
A total of 393 alleles were detected from the 251 sorghum genotypes, with the number of alleles ranging from 2 (Xcup11) to 24 (Sb5-206) and an average of 10.07 per primer pair (Table 1). The major allele frequency among the SSR markers ranged from 0.13 (Sb5-206) to 0.92 (Xtxp339 and mSbCIR246), with an average of 0.50. The observed heterozygosity was lowest (H o= 0.00) for ISEP0310 and mSbCIR223 and highest (H o= 0.15) for Xtxp136. The expected heterozygosity, also referred to as gene diversity, varied from 0.14 (Xtxp339) to 0.93 (sb5-206). The PIC estimates of the SSR markers studied ranged from 0.14 (Xtxp339) to 0.92 (Sb5-206). Among these markers, nine SSRs (Xtxp012, Xtxp295, Xtxp265, Xtxp141, Sb6-84, Xtxp015, Xtxp23, Sb5-206 and mSbCIR238) had a very high (>0.8) PIC value.
Table 1 Features of the SSR markers used in this study
A, major allele frequency; N, number of alleles; H e, gene diversity; H o, observed heterozygosity; PIC, polymorphism information content.
Nei's genetic distance and F statistics
For understanding the level of genetic differentiation and structure among different races, geographical origin and the elite sorghum groups, Nei's genetic distance and Wright's FST statistics were estimated. The 44 elite genotypes were classified based on their adaptation such as rainy and post-rainy seasons for comparing the diversity and structure with different sorghum races/geographical groups. Among the different basic and hybrid races, the highest pairwise Nei's genetic distance was observed between kafir-caudatum and durra-bicolor (0.31) and the lowest between Guinea-caudatum and caudatum (0.08) (Table 2). When the races were compared with the elite working groups, the rainy sorghum group was found to be highly diverse with the race kafir-caudatum (0.28) and durra-bicolor (0.25) and the less diverse with guinea-caudatum (0.12). The post-rainy sorghum group was also found to be highly diverse with the race kafir-caudatum (0.37) followed by the rainy sorghum group (0.26). The post-rainy sorghum group was more closely associated with durra (0.06) and durra-bicolor (0.06).
Table 2 Pairwise Nei's genetic distance (above the diagonal) and FST estimates (below the diagonal) among the different sorghum races and elite sorghum working groups
OTU, operational taxonomic units.
The pairwise FST was also estimated among the different races and working groups. Among the races, the highest differentiation was recorded between durra and kafir-caudatum (0.42) and caudatum with kafir-caudatum (0.42). The lowest genetic differentiation was observed between bicolor and caudatum (0.001) and caudatum-bicolor (0.001). Comparing the races with the elite groups identified the post-rainy sorghum types to be highly differentiated from kafir-caudatum (0.51) and least differentiated from durra-bicolor (0.01) and durra (0.10). The rainy types were highly differentiated from the kafir-caudatum race (0.47) and least differentiated from the guinea-caudatum race (0.15).
Similarly, Nei's pairwise genetic distance comparisons were made among the different geographical groups and elite groups (Table 3). The highest distance of 0.10 was observed between Africa and East Asia, East Asia with North America and South Asia and the lowest distance between North America and Africa (0.03). The rainy sorghum types were highly diverse with East Asia (0.24) and South-West Asia (0.23). Similarly, the post-rainy sorghum types were highly diverse with East Asia (0.19) and the rainy sorghum group (0.26). Among the various pairwise combinations, the post-rainy and rainy sorghum types were highly diverse (0.26). The pairwise FST comparisons also revealed that the African group was highly differentiated from the East Asian group (0.34), and showed low differentiation between the African and North American groups (0.02). Greater differentiation was observed between the rainy and post-rainy groups (0.37).
Table 3 Pairwise Nei's genetic distance (above the diagonal) and FST estimates (below the diagonal) among the different sorghum geographical groups
OTU, operational taxonomic units.
Admixture model-based population clusters
Genetic structure analysis using the STRUCTURE program was run for the subgroup numbers (K) varying from 2 to 10 for each group. The ΔK method showed the highest peak at K= 2. This provided support for the presence of two genetically distinct clusters. Genotypes with membership coefficients >0.80 were classified into respective groups (groups I and II), while the remaining accessions were classified as the admixture group. Based on this, group I consisted of 93 accessions, group II with 86 accessions while 72 genotypes were considered as admixtures. Group I was mostly dominated by accessions belonging to the races kafir (18), caudatum (11), kafir-caudatum (7), guinea-caudatum (11), Guinea (5), durra (4) and 23 elite genotypes. Coding of accessions based on place of origin revealed that most of the African accessions (56) occupied group I.
Accessions from the races durra (16), caudatum-bicolour (14), durra-caudatum (8), Guinea-caudatum (8), durra-bicolor (7), bicolor (4), caudatum (7), caudatum-bicolor (5) and 17 elite genotypes mainly constituted group II. The accessions belonging to East Asia and South-West Asia strictly occupied group II, and few accessions from these regions were also found in the admixture group. The admixture group included majority of accessions from guinea (13), caudatum (16), bicolor (13) and caudatum-bicolor (8), durra (5), Guinea-caudatum (4) and four elite lines. The caudatum accessions, apart from representing the admixture group, also occupied a good number of genotypes in groups I and II. Similarly, the durra accessions occupied predominantly in group II and in the admixture group.
Clustering of mini-core and elite sorghum genotypes
Neighbour-joining analysis clustered the 251 accessions into five major groups (Fig. 1). The African genotypes showed wide spread clustering indicating high diversity among them. Most of the accessions from South-West Asia and East Asia formed separate groups. The elite genotypes of India grouped based on the season of their adaptation such as rainy and post-rainy, and further revealed narrow diversity among them. Comparing the race and geographical groups, the strongest clustering was observed based on geographical origin, except Africa which showed widespread clustering.
Fig. 1 Neighbour-joining tree showing the relationship among the elite sorghum genotypes of India with mini-core accessions as revealed from SSR analysis. Red colour indicates elite rainy sorghum genotypes of India, while blue colour indicates elite post-rainy sorghum genotypes.
Group I was mostly constituted by the accessions belonging to the race kafir and their intermediates, and showed a strong association between them. Accessions belonging to the rest of the races formed a strong subgroup in group I. The maintainer lines 27B and IMS 9B of the Indian rainy sorghum hybrid breeding programme also occupied a subgroup in group I. In addition, the restorer line CS 3541 of the hybrids CSH 5 and CSH 9 also occupied group I. The accessions IS 29 239 (kafir), IS 29 468 (guinea-caudatum), IS 12 697 (bicolor) and IS 21 645 (guinea) were highly diverse germplasm accessions in group I.
Group II was predominantly occupied by the rainy sorghum genotypes of the elite Indian group. The rainy sorghum genotypes included the hybrid parental lines (maintainer, restorer, varieties and advanced breeding lines). The restorer lines (RS 627, RS 673, NR 486 and AKR 354), varieties (SPV 1616, CSV 13, CSV 15 and SPV 462) and advanced breeding lines (BN 527, BN 538, BN 534, BN 535 and BN 562) were closely associated and formed a subgroup in group II. The nature of clustering revealed narrow diversity within the varieties, restorer lines and advanced breeding lines. Few germplasm accessions from the races caudatum, durra and bicolor and their intermediates belonging to South Asia, North America and Africa formed a strong subgroup in group II. AKR 150, the restorer line of the early duration rainy sorghum hybrid CSH 14, was also a member of the subgroup. The germplasm lines IS 27 786 (durra-bicolor), IS 20 697 (caudatum) and IS 2379 (caudatum) were divergent members of group II.
Group III was constituted by a majority of accessions from Africa and few accessions from North America, South Asia and South-West Asia, and were predominated by the basic and hybrid races of guinea and caudatum and bicolor. This group was one among the divergent groups as none of the genotypes from the elite Indian sorghum group was found to be associated with this group. The accessions IS 12 804 (bicolor) and IS 8348 (durra) from South-West Asia were highly diverse in this group.
Group IV was the smallest group and included nine accessions. All the accessions were from Africa, except IS 20 816 from North America. IS 23 644 from the guinea race was the divergent accession in this group.
Group V was predominated by the post-rainy sorghum genotypes of the Indian breeding programme. The post-rainy sorghum included the recently released varieties (Phule chitra, Phule vasudha, Phule amrutha, CSV 18R, CSV 216R and PKV ashwini) and advanced breeding lines (RSSGV 17, RSSGV 6, RSSGV 43, RSSGV 44) of post-rainy season adaptation. The nature of clustering revealed narrow diversity among the post-rainy sorghum genotypes. Few germplasm accessions from South Asia showed a strong association with the post-rainy sorghum group. The post-rainy sorghum group also showed a close association with the accessions from South-West Asia. RSSGV 43 was diverse in this group and found to be associated with the germplasm lines of the durra and durra-bicolor group. IS 2389 and IS 2872 were substantially diverse. Although the accessions from South-West Asia were clustered in group V, they showed high diversity and formed a separate subgroup. Most of the accessions from East Asia formed a strong subgroup and were found to be associated with group V. Among the East Asian accessions, IS 12 735 and IS 30 466 from the caudatum bicolor race were found to be the divergent members.
Discussion
SSR characteristics and diversity statistics
To evaluate the informativeness and efficiency of the SSR markers in the analysis of genetic diversity as well as in the assessment of population differentiation, the number of alleles, PIC value, observed heterozygosity and gene diversity for each of 39 SSR loci were estimated. A very high number of alleles (393) with an average of 10.07 per locus were obtained in our study. Similar results were obtained by Deu et al. (Reference Deu, Sagnard, Chantereau, Calatayud, Herault, Mariac, Pham, Vigouroux, Kapran, Traore, Mamadou, Gerard, Ndjeunga and Bezancon2008) who reported 292 alleles with an average of 10.43 using 28 SSR markers in 484 Nigerian cultivars and landraces using Li-Cor automated sequencers. Thudi and Fakrudin (Reference Thudi and Fakrudin2011) noted 228 alleles using 30 microsatellites among 42 sorghum genotypes from India with 6% PAGE assay. Folkertsma et al. (Reference Folkertsma, Rattunde, Chandra, Raju and Hash2005) reported 123 alleles using 21 microsatellites in 100 sorghum accessions belonging to the guinea race from ten African countries including India, using an ABI 3700 automatic DNA sequencer. Likewise, Ghebru et al. (Reference Ghebru, Schmidt and Bennetzen2002) detected a total of 208 alleles with an average of 13.9 alleles/locus using 15 microsatellites in 28 accessions of Eritrean landraces using an ABI 377 prism sequencer. The higher number of alleles detected in the present study is primarily attributed to the diverse genetic materials used (Bhosale et al., Reference Bhosale, Stich, Rattunde, Weltzien, Haussmann, Hash, Melchinger and Parzies2011) as they represent all five major basic races and ten intermediate races and are also geographically diverse. In addition, the number of SSR markers used in our study is quite high compared with those reported in the aforementioned studies. Number of alleles is also influenced by the techniques used for size fragmentation of PCR amplicons. PAGE gives more appropriate results as they can resolve minor base-pair (2–3 bp) differences. However, advanced techniques such as capillary electrophoresis or DNA sequencer are currently the choice for genotyping as they can resolve single base-pair differences. In the present study, nine SSR markers (Xtxp012, Xtxp295, Xtxp265, Xtxp141, Sb6-84, Xtxp015, Xtxp23, Sb5-206 and mSbCIR238) showed a very high PIC value (>0.80), indicating a very high discriminating ability of these markers. This is in contrast to our earlier studies (Ganapathy et al., Reference Ganapathy, Gomashe, Rakshit, Prabhakar, Ambekar, Ghorade, Biradar, Saxena and Patil2012; Rakshit et al., Reference Rakshit, Gomashe, Ganapathy, Elangovan, Ratnavathi, Seetharama and Patil2012), in which we obtained PIC values of 0.36 and 0.49, respectively. Botstein et al. (Reference Botstein, White, Skolnick and Davis1980) indicated the PIC value >0.5 as a highly polymorphic locus. Similar results showing PIC values >0.5 (i.e. 0.83 and 0.61) in sorghum were reported by Fernandez et al. (Reference Fernandez, de Haro, Distefano, Martinez, Lia, Papa, Olea, Tosto and Hopp2013) and Adugna (Reference Adugna2014). These markers can be used for effectively partitioning the genetic diversity in diverse sorghum genotypes.
Population structure
The knowledge on population structure is important to determine the degree of genetic relatedness among the individual accessions. For determination of population structure among the 251 genotypes, Bayesian STRUCTURE analysis, Wright's FST statistic and Nei's genetic distance were used. The STRUCTURE analysis revealed ΔK= 2, indicating two major groups (groups I and II), and the rest was considered as admixtures (data not shown). Group I was dominated by accessions belonging to the races kafir, kafir-caudatum, Caudatum and guinea-caudatum and 21 genotypes belonging to the rainy sorghum group. The majority of germplasm accessions belonging to the above-mentioned races were of African origin. Morris et al. (Reference Morris, Ramu, Deshpande, Hash, Shah, Upadhyaya, Riera-Lizarazu, Brown, Acharya, Mitchell, Harriman, Glaubitz, Buckler and Kresovich2013) reported that the kafir race from Southern Africa forms the strongest subdivision relative to other races. Wang et al. Reference Wang, Upadhyaya, Burrell, Sahraeian, Klein and Klein(2013a) also reported the strongest clustering by geographical origin within the accessions belonging to Southern African countries. The races durra, durra-caudatum, durra-bicolor, Guinea-caudatum and 17 elite genotypes of post-rainy season adaptation mainly constituted group II (data not shown). The majority of accessions constituting group II were from South Asia, South-West Asia and East Asia. Morris et al. (Reference Morris, Ramu, Deshpande, Hash, Shah, Upadhyaya, Riera-Lizarazu, Brown, Acharya, Mitchell, Harriman, Glaubitz, Buckler and Kresovich2013) reported that sorghum accessions belonging to the durra race from warm desert climates of the horn of Africa, Sahel, Arabian Peninsula and West Central India formed a distinct cluster, and demonstrated that the accessions from this race could be further differentiated based on their origin. The admixture group included majority of accessions from the races guinea, caudatum, bicolor and caudatum-bicolor. This indicated that these races are highly variable. Morris et al. (Reference Morris, Ramu, Deshpande, Hash, Shah, Upadhyaya, Riera-Lizarazu, Brown, Acharya, Mitchell, Harriman, Glaubitz, Buckler and Kresovich2013) and Wang et al. (Reference Wang, Upadhyaya, Burrell, Sahraeian, Klein and Klein2013a) further reported that accessions from the guinea race formed several distinct groups but were limited by their geographical origin. The Bayesian STRUCTURE analysis revealed a strong genetic structure between the races belonging to groups I and II.
More importantly, the STRUCTURE analysis could classify the 44 elite genotypes into rainy and post-rainy adaptive groups. The results also corroborated with the results of Wright's FST statistic and Nei's genetic distance estimates. The observed genetic structure between the rainy and post-rainy sorghum groups of India is supported by the fact that rainy season sorghum group is rarely used as the source material for the genetic improvement of post-rainy sorghum genotypes and vice versa. The presence of a strong genetic structure indicates that these two groups are reproductively and genetically isolated from each other. Two subpopulations derived from the original population are said to be isolated from each other to allow the selection and fixation of unique alleles, which will then account for divergence and genetic structures among populations (Hartl and Clark, Reference Hartl and Clark1997). Similar results were obtained by Ganapathy et al. (Reference Ganapathy, Gomashe, Rakshit, Prabhakar, Ambekar, Ghorade, Biradar, Saxena and Patil2012). Among the rainy and post-rainy adapted sorghum groups, the post-rainy sorghum types possess desirable grain yield and quality attributes that suit consumer needs (Reddy et al., Reference Reddy, Reddy, Sadananda, Dinakaran, Ashok Kumar, Deshpande, Srinivasa Rao, Sharma, Sharma, Krishnamurthy and Patil2012). However, the post-rainy sorghum types cannot be cultivated during the rainy season as they are photosensitive. The FST statistics also revealed that the rainy and post-rainy groups are highly differentiated from the race kafir-caudatum. Among the geographical regions, both rainy and post-rainy groups were highly differentiated from the East Asian accessions. Therefore, accessions from the above-mentioned race and geographical regions could be potential candidates for the diversification of the Indian breeding lines.
Genetic clustering of the mini-core and elite sorghum genotypes
Genetic clustering of the accessions revealed substantial variation among the mini-core and the elite genotypes of rainy and post-rainy sorghum of India. Both race and geographical origin had a significant influence on the pattern of genetic diversity and genetic structure in sorghum. The neighbour-joining analysis classified the 251 accessions into five major groups; however, substantial diversity existed within each of the five major groups. Greater diversity existed among the African accessions as evidenced from widespread clustering. Folkertsma et al. (Reference Folkertsma, Rattunde, Chandra, Raju and Hash2005) reported greater diversity within African accessions, especially between accessions from semi-arid and Sahelian Africa and those with West Africa, Southern Africa and East Africa. The greater diversity among the African groups is due to the presence of all the basic and intermediate races. In the present study, the 44 elite genotypes, which represented the diversity between the rainy and post-rainy sorghum groups of India, were compared with the mini-core collections to understand the extent and pattern of diversity in the cultivated sorghum. The study facilitated the identification of diverse accessions from various basic and intermediate races and/or diverse geographical origin for future genetic improvement of sorghum in India.
Sorghum in India is cultivated during rainy and post-rainy seasons with cultivars specifically adapted to each season. Race-wise exploitation of sorghum has been a major component in the breeding of both rainy and post-rainy sorghum groups in India. Both varietal and hybrid development are the major part of genetic improvement of rainy sorghum genotypes. Majority of the rainy sorghum genotypes are developed from the races caudatum, kafir and their intermediate types (Audilakshmi et al., Reference Audilakshmi, Aruna and Kiran2003). Heterosis is best exploited in the rainy season; however, for the rapid development of hybrid parental lines and varieties, breeders have recently started crossing one advanced variety with the other. This results in the narrow genetic base of the breeding population, which was also evident from the present study in which close grouping was observed among the rainy sorghum genotypes. Narrow diversity was observed between the rainy sorghum restorer lines (RS 627, RS 673, NR 486 and AKR 354), varieties (SPV 1616, CSV 13, CSV 15 and SPV 462) and advanced breeding lines (BN 527, BN 538, BN 534, BN 535 and BN 562). The narrow diversity between the restorers and the varieties is due to high gene flow between these groups, which share a common gene pool. Tall lines with complete fertility restoration and longer pollen shedding duration are preferred as restorer lines, while those restorer lines with high yielding ability and superior grain quality traits are used as varieties for commercial cultivation (Andrews et al., 1997; Rooney and Smith, Reference Rooney, Smith, Smith and Frederiken2000). Within the restorer lines, a close association was observed, except AKR 150 and C 43, indicating narrow diversity. This necessitates the need for diversifying the restorer group for further maximizing heterosis in rainy sorghum hybrid breeding. From the cluster analysis, the maintainer lines grouped apart from the restorer group but occupied the same major group. This distinct grouping of maintainers and restorers is largely due to the fact that separate breeding programmes are being followed for seed parents and their restorers, and, more importantly, a separate gene pool is being maintained to maximize the level of heterosis (Rooney and Smith, Reference Rooney, Smith, Smith and Frederiken2000). The maintainer lines 27 B, IMS 9B and 296 B were substantially diverse from the rest of the maintainer lines. The high diversity of 27 B is reflected by high heterosis level in its hybrid CSH 16 (27 A × C 43) released during 1997, being still the most popular hybrid, and occupies a major proportion of the rainy sorghum area in the country. Similarly, the seed parent 296 B is highly heterotic and has been successfully used as the seed parent for the development and release of rainy sorghum hybrids CSH 9, CSH 10, CSH 11, CSH 12 and CSH 13R. CSH 13R is a successful hybrid in terms of its yielding ability both for grain and fodder, but due to the lack of synchronization in flowering between the female parent (296 A) and the male parent (RS 29), the hybrid has not become popular. In the present study, 296 B became associated with the post-rainy group as it includes durra as one its parent. Recently in India, the yields of rainy sorghum hybrids and varieties have stagnated due to the narrow genetic base utilized in breeding programmes, and there is an urgent need to diversify the genetic base for breaking the yield plateau, which is possible by pyramiding genes belonging to different yield components. Studies have indicated that the guinea race contributes significantly to a higher mean and heterosis for grain yield (Reddy and Prasada Rao, Reference Reddy and Prasada Rao1993), especially for yield enhancement in rainy sorghum genotypes. Aruna and Audilakshmi (Reference Aruna and Audilakshmi2008) reported that germplasm lines from guinea, kafir and inter-races are good sources for different yield components in addition to the caudatum race. In the present study, the accessions IS 29 239 (kafir), IS 29 468 (guinea-caudatum), IS 12 697 (bicolor), IS 21 645 (guinea), IS 12 804 (bicolor), IS 23 644 (guinea), IS 29 239 (kafir), IS 29 468 (guinea-caudatum), IS 12 697 (bicolor), IS 21 645 (guinea), IS 20 697 (caudatum), IS 2379 (caudatum), IS 2389 (kafir), IS 30 466 (caudatum-bicolor) and IS 12 735 (caudatum-bicolor) were highly diverse from the neighbour-joining analysis compared with the rest of the germplasm accessions, and therefore they can be suitable candidates for the genetic improvement of rainy sorghum genotypes.
Post-rainy sorghum types in India occupy a major proportion of the total sorghum area in the country. Grains of the post-rainy sorghum group are best utilized for food, especially in the form of rotis/bhakris, and therefore in addition to yield, grain quality is also an important attribute while improving yield in this group. Post-rainy sorghum is predominantly occupied by the varieties, and heterosis is least exploited. Two hybrids (CSH 13R and CSH 15R) were bred for the post-rainy sorghum group but did not become popular. Genetic improvement of post-rainy sorghum in India utilizes landrace M35-1 or a similar local durra race as one of the parents, which has resulted in the narrow diversity of post-rainy sorghum. Attempts were also made to introgress heterosis from rainy sorghum to post-rainy cultivars; however, significant progress could not be achieved due to the problems associated with grain quality and adaptation in such crosses. The M35-1 or local durra has superior grain quality (bold grain and grain lustre) and are adapted to adverse soil conditions, and therefore they are routinely used in breeding programmes. Except CRS 14, close grouping was observed among the post-rainy varieties and the breeding lines of the post-rainy season adaptation, indicating narrowing down of the genetic base of the post-rainy sorghum programme. RS 585, a restorer line of the hybrid CSH 15R, was also closely associated with the post-rainy varieties. RS 585 is bred using genotypes of both rainy and post-rainy types. SPV 1758, an elite line with high protein digestibility, was also closely associated with the post-rainy group. As many of the varieties were evolved from the selection or through the hybridization of locally available landraces, it has now become imperative that the exotic types from durra and its intermediate races could be tested for the genetic improvement of yield, quality and adaptation to drought in post-rainy sorghum genotypes. IS 25 836, IS 8348 and IS 2389 were identified as highly diverse accessions from the race durra, and could be potential candidates for the genetic improvement of post-rainy sorghum types.
The pattern of genetic diversity evidenced from the present study provided more detailed insights into the genetic utilization of the germplasm lines as well as their conservation. Sharma et al. (Reference Sharma, Upadhyaya, Manjunatha, Rao and Thakur2013) utilized the sorghum mini-core collection for studying resistance sources to foliar diseases such as anthracnose, leaf blight and rust. Apart from that, it was utilized for mapping traits such as plant height and maturity (Upadhyaya et al., Reference Upadhyaya, Wang, Gowda and Sharma2013a), agroclimatic traits (Morris et al., Reference Morris, Ramu, Deshpande, Hash, Shah, Upadhyaya, Riera-Lizarazu, Brown, Acharya, Mitchell, Harriman, Glaubitz, Buckler and Kresovich2013), leaf rust and grain mould tolerance (Upadhyaya et al., Reference Upadhyaya, Wang, Sharma and Sharma2013b), and anthracnose (Upadhyaya et al., Reference Upadhyaya, Wang, Sharma and Sharma2013c). In an effort to utilize the mini-core collection for the genetic improvement of rainy and post-rainy sorghum types, the mini-core accessions were also evaluated for various yield and quality traits. The germplasm lines IS 4092, IS 23 521, IS 23 579, IS 23 586, IS 23 590 and IS 26 222 recorded high yield and had 100-seed weight more than 4.0 g. In addition, the lines IS 2864, IS 13 971, IS 14 090 and IS 17 941 recorded high protein digestibility (>70%), which is an important quality trait for improving the food value of sorghum. The diverse and trait-specific germplasm identified in the present study could be utilized in a series of diallel programmes for broadening the genetic base and subsequent yield and quality enhancement of rainy and post-rainy sorghum types in India.
Supplementary material
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/S1479262115000441
Acknowledgements
The authors thank Dr Upadhyaya, Principal Scientist, ICRISAT, Dr Elangovan, Principal Scientist, Directorate of Sorghum Research for providing the seed materials of the mini-core collections for the present study. This work was partially carried out by the financial assistance under the NAIP project on ‘Bioprospecting of genes and allele mining for abiotic stress tolerance’ (Project code 4151).
