RFLP analysis of cytoplasmic male-sterile lines of pigeonpea [Cajanus cajan (L.) Millsp.] developed by interspecific crosses

Total DNA from three putative cytoplasmic male sterile (CMS) progenies derived from crosses between the wild species Cajanus sericeus and the cultivated species Cajanus cajan, five C. cajan, one accession of C. sericeus and two genetic male sterile lines of pigeonpea were compared for their RFLP patterns using maize mitochondrial DNA (mtDNA) specific probes. Three putative cytoplasmic male sterile (CMS) progenies from the multiple cross genome transfer of pigeonpea lines (CMS 7–1, CMS 12–3, and CMS 33–1) showed hybridization patterns identical to that of C. sericeus when DNA was digested with EcoRI and HindIII and probed with maize mtDNA clones. The results suggested that these putative CMS progenies have the mitochondria of the female wild species parent. The hybridization patterns of the three male parental lines used in the development of the CMS progenies were similar in all the restriction enzyme-probe combinations except HindIII-atp6. The genetic male sterile lines, MS Prabhat and QMS 1 differed from each other in their hybridization pattern. The genomic DNA hybridization pattern of HindIII digested DNA from ICPL 87 differed from the other pigeonpea lines when probed with the maize mtDNA clones. The cluster analysis of the hybridization data suggested the occurrence of variation in the mitochondrial genome even among the cultivated species. This revised version was published online in July 2006 with corrections to the Cover Date.     

A mechanism for inhibiting cross-fertilization in pigeonpea (Cajanus cajan (L.) Millsp.)

Summary Natural out-crossing imposes considerable costs and inefficiencies in breeding, evaluation and commercialization of pigeonpea (Cajanus cajan (L.) Millsp.). This note reports identification of a modification of floral morphology which inhibits cross-fertilization. Floral morphology and possible mechanisms of action of this character are discussed.On leave from International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). Hyderabad, India.     

First information on heterotic groups in pigeonpea [Cajanus cajan (L.) Millsp.]

To break the decades-old yield barrier in pigeonpea [Cajanus cajan (L.) Millsp.] a hybrid breeding technology was successfully developed and the first two hybrids were recently released in India. In order to produce heterotic hybrid combinations, the first logical step is the identification and selection of genetically diverse parents with favorable alleles. In this context, the concept of classifying hybrid parents into different heterotic groups was developed and successfully used in maize and later adopted in other crops. Since hybrid technology in pigeonpea is new, the authors have made the first attempt to identify heterotic groups using SCA effects of 102 crosses generated from line × tester mating and evaluated them at four locations. Based on the performance of hybrids in terms of SCA effects, seven heterotic groups were constituted. Besides this, a scheme to use this information in breeding high yielding hybrids with specific or wide adaptation is also discussed herein. Genetic diversity between lines and tester showed positive association with the heterotic pools generated on the basis of SCA.     

Inheritance and molecular mapping of restorer‐of‐fertility (Rf) gene in A2 hybrid system in pigeonpea (Cajanus cajan)

Inheritance of fertility restorer gene in pigeonpea was studied using F₂ and BC₁F₁ populations derived from cross AL103A × IC245273. It was found to be controlled by single dominant gene. Out of 228 SSR primer pairs, 33 primer pairs showed parental polymorphism, while four primers were found polymorphic in bulk segregant analysis (BSA). These four primers viz., CcM 1615, CcM 0710, CcM 0765 and CcM 1522 were used for genotyping of F₂ population and were found to be placed at 3.1, 5.1, 28.1 and 45.8 cM, respectively. Two of them, CcM 1615 and CcM 0710, evinced clear and unambiguous bands for fertility restoration in F₂ population. The Rf gene was mapped on linkage group 6 between the SSR markers CcM 1615 and CcM 0710 with the distances of 3.1 and 5.1 cM, respectively. The accuracy of the CcM 1615 was validated in 18 restorers and six maintainer lines. The marker CcM 1615 amplified in majority of male restorer lines with a selection accuracy of 91.66%.     

Development of genomic simple sequence repeat markers from an enriched genomic library of grass pea (Lathyrus sativus L.)

Simple sequence repeat (SSR) or microsatellite markers are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker‐assisted selection and breeding. In this study, a SSR genomic enriched library was developed in Lathyrus sativus (grass pea) by affinity capture of restriction fragments to biotinylated microsatellite oligonucleotides. About 400 randomly selected clones were sequenced, and SSRs were present in approximately 30% of them. Clones contained 75%, 9% and 16% of simple, interrupted and compound SSRs, respectively. Of the 10 SSRs tested, 7 primer pairs produced clearly distinguishable DNA banding patterns. Successively, SSR primer pairs were successfully tested to reveal polymorphism in a set of four different grass pea germplasm accessions. The transferability of SSR markers was high among three related species of Lathyrus, namely Lathyrus cicera, Lathyrus ochrus and Lathyrus tingitanus, and the legume crop, Pisum sativum. These results indicate that the novel SSR markers are informative and will be useful and convenient for genetic analysis in grass pea and related species.     

Identification of two RAPD markers genetically linked to a recessive allele of a Fusarium wilt resistance gene in pigeonpea (Cajanus cajan L. Millsp.)

Summary Fusarium wilt (Fusarium udum Butler) is a soil borne disease of pigeonpea which causes substantial yield losses. The disease can occur at any stage of plant development, from the young seedling to the pod filling stage. Though resistance is simply inherited, transfer to locally adapted cultivars has been difficult due to linkage drag and difficulty in accurate phenotyping, except in sick plots. An attempt was made to identify RAPD markers associated with wilt phenotype by using F2 populations derived from contrasting parents; GSl (susceptible) ‘ICPL87119 (resistant) and GS1’ ICP8863 (resistant). Parents and F₂s were grown in a national _Fusarium sick-plot at Gulbarga, India and phenotyped as resistant or susceptible during the entire crop growth period. In both the crosses, resistance to wilt segregated as a monogenic dominant character. DNA samples extracted from sick plot grown, early seedling stage plants of parents and 254 F₂ plants of GS1 × ICPL87119 were held separately for marker identification. PCR reactions using 340 random decamer primers with genomic DNA of parents resulted in detection of 45 polymorphic amplicons from 39 primers. PCR testing of bulked DNA from subsets of resistant and susceptible plants revealed the presence of two amplicons at 704 bp and 500 bp (OPM03704 and OPAC11500) with susceptibility. Analysis of individual F₂ plants showed a segregation ratio of 3: 1 for the presence: absence of the amplicon in both crosses. Considering the wilt reaction and susceptibility-linked RAPD marker, it was possible to deduce genotype of every F₂ plant and the genotypic ratio for wilt reaction was 1RR: 2Rr: 1rr, as expected.     

Genetic diversity and structure of landrace of lablab (Lablab purpureus (L.) Sweet) cultivars in Thailand revealed by SSR markers

Lablab (Lablab purpureus (L.) Sweet) is a legume crop widely cultivated in tropical and subtropical regions of Africa and Asia. In this study, we assessed genetic diversity and population structure of 299 individuals of subspecies purpureus and bengalensis of lablab from Thailand using 13 simple sequence repeat (SSR) markers. The SSR markers detected only 34 alleles in total with a mean of 2.6 alleles per locus. Overall gene diversity was 0.360. Gene diversity (H_E) and allelic richness (A_R) in different geographic regions was comparable. Similarly, both H_E and A_R between subspecies purpureus and bengalensis were similar. STRUCTURE and neighbor-joining (NJ) analyses revealed that the 299 individuals were clustered into two major groups. In contrast, principal coordinate analysis (PCoA) revealed admixture of the lablab germplasm. STRUCTURE, NJ and PCoA analyses also revealed that the subspecies purpureus and bengalensis are not genetically differentiated. Although the number of individuals from the west of Thailand was small and all of them were collected from the same province, they possessed comparable gene diversity with those from the other geographic regions. These results demonstrated that there is moderately low genetic diversity of lablab in Thailand and the west of the country possesses high diversity of lablab.     

Identification of stable restorers and genetics of fertility restoration in late‐maturing pigeonpea [Cajanus cajan (L.) Millspaugh]

A total of sixty‐six germplasm lines were crossed with five CMS lines, where two belong to A₄ cytoplasm, while other three belong to A₂ cytoplasm. On the basis of pollen fertility test as well as good pod setting, of 330 hybrids, 34 restorer lines were observed in ICPA 2043 and 19 in ICPA 2092. Thirteen germplasm lines restored fertility in both the A₄ CMS lines, viz. ICPA 2043 and ICPA 2092; however, none of the lines restored fertility in A₂ CMS lines. For confirmation of result, restoration competence of identified lines tested subsequently 2 years at two different temperatures. The segregation patterns for fertility restoration studied in F₂ and BC₁F₁ generations of selected ten crosses. Six crosses indicated the involvement of two major genes with recessive epistasis, three crosses confirmed dominant epistasis, and one cross indicated the involvement of duplicate recessive epistasis. The obtained results from this study will hasten the future three‐line breeding programme and lead the hybrid technology to the farmers' field with the better exploitation of CMS lines.     

Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.     

Evaluation of Upland Rice (Oryza sativa L.) Genotypes for Intercropping with Pigeon Pea (Cajanus cajan [L.] (Mill sp.))

Eleven upland rice genotypes of varying growth duration and plant stature were evaluated in two cropping systems: monocrop and intercrop, with pigeon pea cv. U pas 120, in order to study the effects of intercropping on rice grain yield and its contributing characteristics during 1990 to 1992 wet seasons. Cropping system and cropping system × genotype interaction effects were significant for yield ha⁻¹, panicle weight, panicles m⁻² and spikelet fertility suggesting the need for evaluating and selecting genotypes suitable for intercropping. Rice grain yield reduction in the intercrop ranged from 24.5 % in genotype RR 203-16 to 54.5 % in genotype Aditya. Panicle weight, total dry matter at flowering as well as at harvest, and harvest index were also reduced. Plant height and panicle weight were positively associated with yield in both systems, however, the yield was positively and significantly correlated with spikelets per panicle and spikelet fertility with the intercropping system. The correlation between cropping systems indicated the possibility of simultaneous improvement for these characteristics in monocropping and intercropping.     

Comparative analysis of cultivated melon groups (Cucumis melo L.) using random amplified polymorphic DNA and simple sequence repeat markers

Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to characterize genetic relationships among 46 accessions in two C. melo L. subsp. melo (Cantalupensis, Inodorus) and subsp.agrestis (Conomon, and Flexuosus) groups. Genetic distance (GD) estimates were made among and between accessions in four melon market classes [Galia, Ogen, Charentais, and Shipper (European and U.S. types)] of Cantalupensis, one market class of Inodorus (Cassaba and Honey Dew), one accession of Conomon, and one accession of Flexuosus by employing three GD estimators; simple matching coefficient, Jaccard's coefficient, and Nei's distance-D. Differences detected among 135 RAPD bands and 54 SSR bands (products of 17 SSR primers) were used to calculate GD. Band polymorphisms observed with 21 RAPD primers and 7 SSR primers were important (p =0.01) in the detection of genetic differences. Estimators of GD were highly correlated (p 0.0001; r_s = 0.64 to0.99) when comparisons were made between estimation methods within a particular marker system. Lower correlations (r_s = 0.17 to 0.40) were detected (P > 0.001) between marker systems using any one estimator. The GD of the Conomon and Flexuosus accessions was significantly different (p> 0.001)from the mean GD of all the market classes examined. The mean GD (Jaccard's coefficient) among accessions of Ogen, Galia, Cassaba, Charentais, European shipper, and U.S. shipper groups was 0.11 ± 0.04, 0.33± 0.09, 0.21 ± 0.04, 0.26 ± 0.10, 0.17± 0.05 and 0.22 ± 0.08, respectively. Market classes were distinct (p > 0.001), such that GDs between Galia and other accessions were the largest(mean GD 0.34 to 0.35), and GDs between Ogen and other accessions were the smallest (mean GD 0.29 to 0.30). Contrasts between the U.S. shipper cultivar Top Mark and accessions within any market class was relatively large (mean GD = 0.42 ± 0.06). Empirical estimations of variances associated with each marker type in the accessions examined indicated that, per band, lower coefficients of variation can be attained in the estimation of GD when using RAPDs compared to SSRs. Nevertheless, the genetic relationships identified using these markers were generally similar. The disparity between the analyses of the two markers made may be related to the amount of genome coverage which is characteristic of a particular marker system and/or its efficiency in sampling variation in a population. Results of RAPD marker analysis suggest that 80 marker bands were adequate for assessing the genetic variation present in the accessions examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity and population structure of Indian soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.] revealed by simple sequence repeat markers

Genetic diversity in 90 Indian soybean cultivars was assessed using 45 SSR markers distributed on 20 soybean chromosomes. Forty-five SSR markers generated 232 alleles with an average of five alleles/locus. The observed frequencies of the 232 alleles ranged from 0.01 to 0.94 with an average of 0.19. The polymorphic information content (PIC) value of the SSR markers varied from 0.10 to 0.83 with an average of 0.61 and about 71% markers have a PIC value of >0.5. In this study, 54 rare alleles including 19 genotype specific alleles were also identified. The observed hetrozygosity for SSR markers ranged from 0 to 0.11 with a mean of 0.10. Cluster analysis grouped the 90 soybean cultivars into three major clusters and principal coordinates analysis (PCoA) results were similar to those of the cluster analysis. A combination of eight SSR markers successfully differentiated all 90 soybean cultivars. The population structure analysis distributed the 90 soybean genotypes into two populations with mean alpha (α) value of 0.1873. In AMOVA analysis, proportion of variation within population was high (88%), whereas only 12% occurred among populations. In cluster and structure analyses, most of the genotypes with similar pedigree were grouped together. Soybean cultivars DS228, MACS-13, LSb-1, Hardee, Improved Pelican, and Pusa-24 were the six most genetically distinct cultivars identified. The study reported a moderate genetic diversity in Indian soybean cultivars and findings would be useful to the soybean breeders in selecting genetically distinct parents for a soybean improvement program.     

Exploring genetic diversity for heat tolerance among lentil (Lens culinaris Medik.) genotypes of variant habitats by simple sequence repeat markers

The study was carried out to assess genetic diversity among 119 lentil genotypes grown in different habitats for heat tolerance using morpho‐physiological and reproductive traits and SSR markers. High‐temperature stress was applied at seedling (35/33°C) and anthesis stages (35/20°C) to study the effects on morpho‐physiological and reproductive traits under hydroponic condition, which was compared with non‐stressed and stressed field conditions. A set of 209 alleles were identified by 35 SSR markers among the genotypes. Genetic diversity and polymorphism information content values varied between 0.0494–0.859 and 0.0488–0.844, with mean values of 0.606 and 0.563, respectively. Genotypes were clustered into nine groups based on SSR markers. Morpho‐physiological and reproductive traits under heat stress were found to be significantly different among SSR clusters. These findings suggest that heat adaptation is variable among the genotypes and the tolerant materials can be evolved through hybridization using parents from different clusters with diverse mechanisms of heat tolerance.     

An overview of male-sterility systems in pigeonpea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.]

For commercial development of hybrids the four pre-requisites are; availability of perfect male-sterility system, efficient mass pollen transfer mechanism, hybrid vigor, and the large scale seed production of hybrids for commercialization. The type of male-sterility governs the acceptance of hybrids by farmers. Genetic male-sterility (GMS) system was not accepted by farmers due to the economics of large scale seed production. The major drawback was rouging of fertile counterpart from the female plot, which was time consuming and labor intensive. Cytoplasmic-genic male-sterility (CMS) system usually was a better option for large scale seed production. Hybrid vigor has been utilized in some cereal and vegetable crops. Pigeonpea (Cajanus cajan) displays considerable natural out-crossing and now CMS lines are available with different cytoplasmic backgrounds. This mini-review reports the research on development of CMS lines and CMS-based hybrids in pigeonpea.     

Segregation distortion in doubled haploid lines of barley (Hordeum vulgare L.) detected by simple sequence repeat (SSR) markers

A total of 147 simple sequence repeat (SSR) markers (including86 barley and 61 wheat microsatellite markers) were tested for their segregation in a doubled haploid (DH) and an F₂ population of barley. The DH population consisted of 71 doubled haploid lines, developed from F₁ plants of a cross between Tadmor and WI2291using isolated microspore culture technique. A genetic linkage map consisting of 43 microsatellite markers was constructed using the DH population. Particularly on chromosome 4H microsatellite markers showed distorted segregation ratios. Segregation of DH lines based on molecular markers were compared with segregation of 92 F₂ lines from the same cross. The proportion of loci deviating from the expected monogenic segregation ratios in the DH population was significantly higher (19/43loci, 44%) than in the F₂ population (7/43 loci, 16%). The deviation was biased towards the WI2291 parent alleles. In line with this observation, WI2291 was found to perform better than Tadmor in regenerating green plantlets with the isolated microspore-culture technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

Methodology of generation and characteristics of simple sequence repeat DNA markers in avocado (Persea americana M.)

Summary Generation of Simple Sequence Repeat (SSR) DNA markers was based on the construction of genomic DNA library of avocado (Persea americana M.). The library was screened with the four dinucleotide probes (AG), (AT), (GC) and (CA). Positive clones were sequenced to validate the presence of simple sequence repeats (SSR) and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the simple sequence repeat. Twenty six different pairs of primers which yield a PCR product in the initial screening were synthesized. The SSR A1E11 was found to have eleven alleles while A3F8 has eight alleles. The SSRs in avocado were found to be inherited in a Mendelian fashion.Contribution from the Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel. No. 1335-E, 1994 series.     

Genomewide association analysis of salt tolerance in soybean [Glycine max (L.) Merr.]

Salinity is a common abiotic stress causing soybean [Glycine max (L.) Merr.] yield loss worldwide. The use of tolerant cultivars is an effective and economic approach to coping with this stress. Towards this, research is needed to identify salt‐tolerant germplasm and better understand the genetic and molecular basis of salt tolerance in soybean. The objectives of this study were to identify salt‐tolerant genotypes, to search for single‐nucleotide polymorphisms (SNPs) and QTLs associated with salt tolerance. A total of 192 diverse soybean lines and cultivars were screened for salt tolerance in the glasshouse based on visual leaf scorch scores after 15–18 days of 120 mM NaCl stress. These genotypes were further genotyped using the SoySNP50K iSelect BeadChip. Genomewide association mapping showed that 62 SNP markers representing six genomic regions on chromosomes (Chr.) 2, 3, 5, 6, 8 and 18, respectively, were significantly associated with salt tolerance (p < 0.001). A total of 52 SNP markers on Chr. 3 are mapped at or near the major salt tolerance QTL previously identified in S‐100 (Lee et al., 2014). Three SNPs on Chr. 18 map near the salt tolerance QTL previously identified in Nannong1138‐2 (Chen, Cui, Fu, Gai, & Yu, 2008). The other significant SNPs represent four putative minor QTLs for salt tolerance, newly identified in this study. The results above lay the foundation for fine mapping, cloning and molecular breeding for soybean salt tolerance.     

Application of inter simple sequence repeat (ISSR) polymorphism for detection of sex-specific molecular markers in hop (Humulus lupulus L.)

Summary Hop (Humulus lupulus L.) is dioecious species with female plants of commercial value. During breeding process it is desirable to identify the sex of hop plants at the stage of seedling. Twenty two inter simple sequence repeat (ISSR) primers were screened on female and male hop genotypes of Russian and European origin. Two ISSR primers revealed fragments specific for male plants of hop. Based on the sequences two pairs of primers were designed. These male specific sequence tagged site (STS) markers were tested on male hop accessions of Russian origin and female hop accessions of Russian, European and American origin. A high homology of male specific hop DNA sequences to expressed sequences from EMBL plants EST database was found, most of which code cell wall glycoproteins. The applicability of ISSR-PCR analysis for development of sex molecular markers in hop is discussed.     

Tagging the juvenile locus in soybean [Glycine max (L.) Merr.] with molecular markers

Soybean cultivars carrying the `long juvenile trait' show a delayed flowering response under short day conditions. The incorporation of this character into genotypes of agronomic interest may allow a broader range of sowing dates and latitudes for a single cultivar adaptation. The objective of this work was to identify molecular markers linked to the juvenile locus in soybean. Experiments were carried out using two pairs of near isogenic lines(NILs) differing in the presence of the long juvenile trait, and RAPD markers. Four hundred primers were first screened to find polymorphism associated with the trait. Additional differences between NILs were sought by digesting the genomic DNA with five restriction enzymes. Polymorphic fragments detected between NILs were tested for linkage to the juvenile locus in the corresponding F₂ segregating populations. Marker bc357-HaeIII was linked (χ²L = 46.316) to the juvenile locus with an estimated recombination frequency of 0.13 ± 0.03in one of the genetic backgrounds studied. The fragment was cloned, sequenced and converted into a SCAR marker. Moreover,bc357-HaeIII was used as RFLP probe. Both, SCAR and RFLP generated markers linked to the juvenile locus in the two genetic backgrounds analysed. Results presented in this work can be utilised for both, the localisation of the gene associated with the character and for tagging the juvenile trait in soybean breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.