Genetic diversity analysis of Moroccan lentil (Lens culinaris Medik.) landraces using Simple Sequence Repeat and Amplified Fragment Length Polymorphisms reveals functional adaptation towards agro‐environmental origins

In the absence of previous molecular characterization, we assessed genetic diversity of 53 Moroccan lentil landraces including two local cultivars using simple sequence repeat (SSR) and amplified fragment length polymorphisms (AFLP). Nineteen SSRs yielded 213 alleles, and seven AFLP primer combinations gave 766 fragments of which 422 were polymorphic. Moderate to high genetic variation was observed. Several small groups of landraces were differentiated. Interestingly, one of the smallest groups contained short‐cycle landraces with high early vegetative growth. Landraces in that group were from the dry land location of Abda, where they were likely selected for adaptation to drought and heat stress over centuries. Another group contained two landraces from highland areas that may have been selected for specific adaptation to cold stress. A third group contained one landrace from the Zear region known for its seed quality and has been proposed for the protected designation of origin (PDO) quality mark. Both techniques gave evidence of differentiation of the latter landrace supporting the idea of PDO attribution. Functional grouping according to agro‐environmental origins, cycle duration and early vegetative vigour was observed.     

Harvest index in lentil (Lens culinaris Medik.)

Summary Correlation and path coefficient analyses were conducted to find out the characters associated with grain yield and dry matter production and the ways to improve harvest index in lentil.Correlation studies revealed positive association of harvest index with grain yield but no association with plant dry matter. Grain yield and plant dry matter showed positive correlation with pod number, plant height, and number of primary and secondary branches but negative correlation with 100-seed weight. Generally, the genotypic correlations were in agreement with phenotypic correlations, though the magnitude of the values was higher in the former case. In path analysis, plant height and pod number showed the highest direct effect and, therefore, seem to be the main characters influencing grain yield and plant dry matter. Number of primary and secondary branches, on the other hand, showed negative direct effect on grain yield and plant dry matter. Use of phenotypic or genotypic correlations in path analysis resulted in similar conclusions. It is, therefore, suggested that either phenotypic or genotypic correlations may be used in path analysis with equal efficiency. Based on this study it is suggested to develop tall varieties with good pod bearing but with low number of branches.     

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.     

A new phenotyping technique for screening for drought tolerance in lentil (Lens culinaris Medik.)

Screening for drought tolerance is severely handicapped by the lack of a simple and reliable phenotyping technique. The objective of this study was to develop a new screening technique based on seedling survivability, drought tolerance score, root and shoot length, and fresh and dry weight of roots and shoots of lentil plants exposed to drought under hydroponic conditions. Its effectiveness was compared with two soil culture techniques. The hydroponic technique involved removing 15‐day‐old seedlings of 80 genotypes from the nutrient solution and exposing them to air for 5 h daily for 6 days. Three genotypes received from ICARDA, ‘ILL‐10700’, ‘ILL ‐ 10823’ and ‘FLIP‐96‐51’, showed maximum seedling survivability and minimum reduction in the growth parameters with a drought score of 0.0–0.2 indicating higher tolerance to drought stress, while Indian genotypes ‘JL‐3’, ‘E‐153’ and ‘VL‐507’ showed no seedling survivability and maximum reduction in growth parameters with a drought score of 4.0 indicating low drought tolerance. The results suggest that this new phenotyping technique is effective, rapid and easy for screening a large number of genotypes.     

Inheritance of black testa colour in lentil (Lens culinaris Medik.)

The inheritance of testa (seed coat) colour and interaction of cotyledon and testa colours were studied in seven crosses of lentil (Lens culinaris Medik.) involving parents with black, brown, tan or green testa and with orange, yellow or dark green cotyledons. Analysis of F₂ and F₃ seed harvested from F₁ and F₂ plants, respectively, revealed that although black testa is dominant over nonblack testa, its penetrance is not complete since both F₁ plants and heterozygous F₂ plants produced varying proportions of seeds with either black or nonblack testa. The F₂ populations of the crosses between parents with brown and tan, as well as brown and green, testa segregated in the ratio of 3 brown : 1 tan and 3 brown : 1 green, respectively, indicating monogenic dominance of brown testa colour over tan or green. The expression of testa colour was influenced by cotyledon colour when parents with brown or green testa are crossed with those having orange or green cotyledons. Thus F₂ seeds from these crosses with a green testa always had green cotyledons and never orange cotyledons. F₂ seeds from these crosses with a brown testa always had orange cotyledons and never green cotyledons. These results suggest diffusion of a soluble pigment from the cotyledons to the testa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of resistance to Fusarium wilt in Indian lentil germplasm (Lens culinaris Medik.)

Summary Three lentil genotypes resistant to Fusarium oxysporum f.sp. lentis viz. Pant L 234, JL 446 and LP 286 were crossed with two susceptible ones. The hybrid plants were all resistant in the eight crosses evaluated. Segregation pattern for wilt reaction in F₂, BC(P₁), BC(P₂) and F₃ generations in field and glasshouse conditions indicated that resistance to Fusarium wilt is under the control of two dominant duplicate genes in Pant L 234 and two independent dominant genes with complementary effects in JL 446 and LP 286. A third dominant gene complementary to the dominant genes in JL 446 and LP 286 is present in two susceptible lines. Allelic tests suggest the presence of five independently segregating genes for resistance. Duplicate dominant genes in Pant L 234 are non-allelic to two dominant genes with complementary effects in LP 286 and JL 446 and the third gene complementary to the two genes in JL 446 and LP 286 in susceptible lines JL 641 and L 9–12. Gene symbols among parental genotypes have been designated.     

Abundance, polymorphism and genetic mapping of microsatellites in oilseed rape (Brassica napus L.)

The objective of the present study was to estimate the abundance and degree of polymorphism of simple sequence repeat (SSR) markers in rapeseed. By screening about 45000 clones of a small inserts library of rapeseed total DNA the abundances of GA/TC and CA/TG simple sequence repeats in the rapeseed genome were estimated to be approximately one repeat every 100 kb and 400 kb, respectively. After sequencing 13 positive clones, primer pairs could be designed for 11 microsatellite loci. Seven of these primer pairs produced reproducible amplification products in a set of 31 rapeseed genotypes, with one pair amplifying two independent products, giving a total of eight amplified loci. The different microsatellite loci displayed between one and three visible alleles. At four loci, additional null alleles were observed. With up to four alleles, polymorphic microsatellite markers show significantly higher allele numbers in rapeseed than restriction fragment length polymorphism (RFLP) markers. Four of the eight microsatellite markers could be mapped on four different linkage groups of an RFLP map of the rapeseed genome.     

Association of isozyme markers with quantitative trait loci in random single seed descent derived lines of lentil (Lens culinaris Medik.)

Summary Polymorphism at isozyme loci was used to locate factors responsible for variation in quantitative traits of lentil. Eight sets of random single seed descent (RSSD) derived lines were developed by advancing individual F₃ plants of interspecific (L. culinaris Medik. × L. orientalis Boiss.) hybrids to the F₆. The RSSD lines in each of the eight sets differed for alleles at 2–8 isozyme loci. In each set, association of isozyme loci with variation in seven quantitative traits (days to flower, days to mature, plant height, biomass, seed yield, harvest index, seed weight) was determined for each pairwise combination of a quantitative trait with a marker locus. Loci affecting variation in all seven quantitative traits were detected by their association with 14 isozyme markers (Aat-c, Aat-m, Aat-p, Adh-1, Fk, Gal-1, Gal-2, Lap-1, Lap-2, Pgd-p, Pgi, Pgm-c, Pgm-p, Skdh). The known position of 10 the 14 isozyme loci on the lentil genetic map was used to mark the genomic regions for possible location of associated quantitative trait loci (QTL). Detected QTL were found to be located in six of the seven linkage groups on lentil genetic map. Regions of the genome represented by linkage groups, 1, 5 and 7 appeared to affect a greater number of traits than other genomic regions represented by linkage groups 2, 3 and 4. Results indicated that the mean expression of quantitative traits at segregating marker locus classes can be used to locate the genetic factors in lentil which influence the behavior of economically important traits.     

Genic microsatellite markers for discrimination of spinach cultivars

A set of simple sequence repeat (SSR or microsatellite) markers was used to discriminate a collection of 33 Spinacia oleracea hybrid cultivars from seven different breeding stations all over the world. All SSR markers were genic microsatellite markers located in coding or non-coding regions of genes of known function. Cluster analysis based on 13 of the SSR markers showed that the spinach hybrids grouped into three clusters. The first two clusters consisted of European spinach types, which were well discriminated according to their origin from different breeding stations. The third cluster was a mixture of Asian as well as European types of spinach. Subclusters in this group did not reflect differences in morphology, earliness or company origin. The data show that genic microsatellites are a powerful tool for discrimination of spinach cultivars.     

Breeding for boron tolerance in lentil (Lens culinaris Medik.) using a high‐throughput phenotypic assay and molecular markers

This study describes the identification of a quantitative trait locus (QTL) in the recombinant inbred line population of ILL2024 × ILL6788 and subsequent validation of associated molecular markers. A high‐quality genetic linkage map was constructed with 758 markers that cover 1,057 cM, with an average intermarker distance of 2 cM. QTL analysis revealed a single genomic region on Lc2 to be associated with B tolerance and accounted for up to 76% of phenotypic variation (Vp). The best markers for B tolerance were assessed for their utility in routine breeding applications using validation panels of diverse lentil germplasm and breeding material derived from ILL2024. A marker generated from the dense genetic map of this study was found to be the most accurate of all markers available for B tolerance in lentil, with a success rate of 93% within a large breeding pool derived from ILL2024. However, given the number of the unrelated lines for which the marker–trait association was not conserved, B tolerance screening is still required at later stages to confirm predicted phenotypes.     

Discovery of a new gene causing dark green cotyledons and pathway of pigment synthesis in lentil (Lens culinaris Medik)

A new gene is reported which functions as a master gene for synthesis of the pigments determining cotyledon colour in lentil. This gene is different from the two earlier reported genes which are responsible for synthesis of yellow (gene Y) and brown (gene B) pigments. Double recessive homozygous condition of these two genes results into loss of both pigments and, consequently, produces light green cotyledons. The new gene, in contrast, produces dark green cotyledons in recessive condition irrespective of the dominance or recessive state of the Y and B genes. It is hypothesized that the new gene for dark green cotyledon colour (Dg) acts at an earlier stage in the biosynthesis of the two cotyledon-specific pigments, which are derived from a common precursor, whose synthesis is blocked when Dg mutates to its recessive condition. This revised version was published online in August 2006 with corrections to the Cover Date.     

QTL mapping of resistance in lentil (Lens culinaris ssp. culinaris) to ascochyta blight (Ascochyta lentis)

Quantitative trait locus (QTL) analysis of ascochyta blight resistance in lentil was conducted using genomic maps developed from two F₂ populations, viz. ILL5588/ILL7537 and ILL7537/ILL6002. Five QTLs for ascochyta blight resistance were identified by composite interval mapping (CIM) across four linkage groups (LG) in population ILL5588/ILL7537. Three QTLs were identified by CIM in population ILL7537/ILL6002 (two in close proximity on LGI and one on LGII). Two of these coincided with regions identified using multiple interval mapping (MIM) and were shown to be conditioned by dominant and partial dominant gene action. Together, they accounted for approximately 50% of the phenotypic variance of disease severity. Comparison between the two populations revealed a potentially common QTL and several common regions that contained markers significantly associated with resistance. This study demonstrated the transferability of QTLs among populations and identified markers closely linked to the major QTL that may be useful for future marker‐assisted selection for disease resistance.     

Inheritance of new genetic markers in lentil (Lens Miller)

Summary We report on the inheritance of 11 morphological markers and 17 isozymes in lentil (Lens culinaris). The monogenic inheritance of 11 morphological markers and 11 isozymes is confirmed. The inheritance of six isozymes (Aco-2, Enp, Est-3, Est-4, Lap-3, and Mdh-m) is reported for the first time in lentil. This brings the total number of described genes in lentil to 78. Cases of disturbed segregation were more frequent than expected by chance. It is suggested that disturbed segregation was in most cases caused by linkage with a piece of chromosome that showed preferential elimination in crosses between Lens culinaris ssp. odemensis and other subspecies. The prevalence of disturbed segregation in crosses with Lens culinaris ssp. odemensis could limit the usefulness of this subspecies in genetic and linkage studies.     

Using molecular markers to pyramid genes for resistance to ascochyta blight and anthracnose in lentil (Lens culinaris Medik)

Ascochyta blight caused by the fungus Ascochyta lentis Vassilievsky and anthracnose caused by Colletotrichum truncatum [(Schwein.) Andrus & W.D. Moore] are the most destructive diseases of lentil in Canada. The diseases reduce both seed yield and seed quality. Previous studies demonstrated that two genes, ral1 and AbR1, confer resistance toA. lentis and a major gene controls the resistance to 95B36 isolate of C. truncatum. Molecular markers linked to each gene have been identified. The current study was conducted to pyramid the two genes for resistance to ascochyta blight and the gene for resistance to anthracnose into lentil breeding lines. A population (F_6:7) consisting of 156 recombinant inbred lines (RILs) was developed from across between ‘CDC Robin’ and a breeding line ‘964a-46’. The RILs were screened for reaction to two isolates (A1 and 3D2) ofA. lentis and one isolate (95B36) ofC. truncatum. χ² analysis of disease reactions demonstrated that the observed segregation ratios of resistant versus susceptible fit the two gene model for resistance to ascochyta blight and a single gene model for resistance to anthracnose. Using markers linked to ral1 (UBC 227₁₂₉₀), to AbR1(RB18₆₈₀) and to the major gene for resistance to anthracnose (OPO6₁₂₅₀),respectively, we confirmed that 11 RILs retained all the three resistance genes. More than 82% of the lines that had either or both RB18₆₈₀ and UBC227₁₂₉₀markers were resistant to 3D2 isolate and had a mean disease score lower than 2.5. By contrast, 80% of the lines that had none of the RAPD markers were susceptible and had a mean disease score of 5.8. For the case of A1 isolate of A. lentis, more than 74% of the lines that carriedUBC227₁₂₉₀ were resistant, whereas more than 79% of the lines that do not have the marker were susceptible. The analysis of the RILs usingOPO6₁₂₅₀ marker demonstrated that 11out of 72 resistant lines carried the marker, whereas 66 out of 84 susceptible lines had the marker present. Therefore, selecting materials with both markers for resistance to ascochyta blight and a marker for resistance to anthracnose can clearly make progress toward resistance in the population. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity and population structure of Indian melon (Cucumis melo L.) landraces with special reference to disease and insect resistance loci

This study was aimed to examine the genetic diversity and population structure of Indian melon landraces with special reference to disease and insect resistance loci. Thirty‐six simple sequence repeat (SSR) markers along with seven markers at disease and insect resistance loci were used for this purpose on a panel of 91 accessions available at Indian Institute of Horticultural Research, Bengaluru, India. Model‐based structure analysis revealed the presence of four groups that were consistent with the results of principal coordinate analysis (PCoA). The delineation of populations was mostly based on geography with improved varieties as a separate group. Ten accessions have been identified to possess beneficial alleles at all the selected disease resistance loci and shall be useful for incorporating multiple disease resistance after phenotypic validation. The results obtained in the current study demonstrate the importance of the Indian melon group as a valuable genetic reservoir and the need to plan strategies for its conservation and utilization in breeding programmes.     

Identification of pre‐ and posthaustorial resistance to rust (Uromyces viciae‐fabae) in lentil (Lens culinaris) germplasm

Lentil (Lens culinaris Medik.) is an important cool‐season legume that can be severely damaged by rust (Uromyces viciae‐fabae (Pers.) Schröt.) in many parts of the world. In this work, a collection of L. culinaris germplasm accessions was screened under growth chamber conditions for response to U. viciae‐fabae. Several sources of incomplete resistance have been identified, both of hypersensitive and of non‐hypersensitive nature. Incomplete hypersensitive resistance was due to a late‐acting host cell necrosis that resulted in an intermediate infection type (IT). Incomplete non‐hypersensitive resistance was due to a hampered cell penetration not associated with host cell necrosis, reducing the number of haustoria per colony and therefore hindering the growth of the colony and reducing and retarding disease development in spite of a high IT.