Polymorphic SSR Markers Research Articles

Development of a reliable Corylus sp. reference database through the implementation of a DNA fingerprinting test.

This DNA fingerprinting test confirmed 195 unique Corylus sp. accessions that were used to build a reference database for identity verification of unknown hazelnut trees from three locations in Ontario. Hazelnut is one of the most profitable tree nuts worldwide. Development of a hazelnut industry in Ontario is urgently required, but economically important cultivars must be genetically verified first in order to meet industry standards. Traditional methods for cultivar identification are largely trait-based and unreliable. In this study, a multiplexed fingerprinting test was modified to allow for hazelnut cultivar discrimination at the DNA level. Fourteen highly polymorphic SSR markers covering the 11 linkage groups of Corylus genome were PCR amplified in multiplex using fluorescent-labelled primers. PCR conditions and primer physical properties were optimized to generate a clear signal for each locus. The 14 SSRs were used to fingerprint 195 unique Corylus accessions collected from the USDA-NCGR. Fragment sizes were subjected to a UPGMA clustering analysis which separated Corylus accessions based on species and geographic origin. For validation purposes, hazelnut leaves from three locations in Ontario were collected for identity verification using this DNA fingerprinting test. As a result, 33.3% of the unknown trees were duplicates of seven distinct genotypes and a small percentage (8.3%) of these were identical to reference Corylus hybrids. These results reflect common mislabelling issues and genotype duplications that can prevent a uniform plant propagation system. Implementation of this test together with the addition of more unique accessions to the reference database will help verification of trueness-to-type of economically important cultivars for the hazelnut industry.

Genetic Mapping and Validation of Quantitative Trait Loci (QTL) for the Grain Appearance and Quality Traits in Rice (Oryza sativa L.) by Using Recombinant Inbred Line (RIL) Population.

Rice grain shape and nutritional quality traits have high economic value for commercial production of rice and largely determine the market price, besides influencing the global food demand for high-quality rice. In order to understand the genetic components of grain appearance traits in paddy, brown, and head rice, 15 traits were evaluated by using 157 recombinant inbred lines (RILs) derived from a cross between two Iranian rice cultivars Ali-Kazemi (A) and Kadous (K). A significant variation was observed and showed transgressive segregation among the RILs. Correlations between the visual appearances of grain traits were studied. A linkage map with 65 polymorphic SSR markers was constructed, which covered 1517.32 cM of the rice genome. A total of seven QTLs were identified on four chromosomes, 1, 6, 9, and 12, associated with four traits, which are explained by the total phenotypic variation of 44.27% and LOD score of 32.77 in 2014 and 2015, respectively. Among these, four QTLs for two traits were consistently flanked by RM23904 and RM24432 on chromosome 9. Single QTL for head grain length (HGL) expressed in both the years on chromosomes 1 and 9. A major QTL for seed weight was detected on chromosome 9, which explained 10.18% of the phenotypic variation. The additive effect of all the QTLs was positively contributed by Ali-Kazemi allele, except one QTL on chromosome 6 (qBGL_6) that showed a negative additive effect being contributed by the Kadous allele. The study also validated the identified QTLs with the polymorphic SSR markers that were previously reported. Novel QTLs were identified on chromosomes 6 and 9, and many of the polymorphic markers were found to be associated with milling processing of grain quality, cooking, and nutraceutical properties of rice by extensive literature and database analysis. Therefore, these validated QTLs and marker information could be utilized in the marker-assisted selection to improve grain appearance and nutritional grain quality traits in rice.

Assessment of genetic diversity and population structure of Magnaporthe oryzae causing rice blast disease using SSR markers

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice in the world. The blast fungus shows a high degree of variability in the field and limited information is available on its genetic diversity and population structure in India. Twenty five polymorphic SSR markers were used to investigate the genetic diversity and population structure of 96 blast isolates collected from Odisha and Chhattisgarh region. The markers yielded a total of 110 alleles and number of alleles per locus varied from 2 to 8. The gene diversity ranged from 0.02 to 0.49 whereas, major allele frequency varied from 0.50 to 0.98. The PIC value varied from 0.02 to 0.37 with a mean value of 0.29. Cluster analysis grouped the blast isolates into four distinct major clusters. The high level of genetic diversity was observed with ninety one haplotypes and nucleotide diversity of 0.293. The principal coordinate and structure analysis identified two genetic clusters of M. oryzae isolates with some degree of distinctness according to affected plant parts. Analysis of molecular variance revealed more genetic variation within populations and less among populations. The outcome of this study would help to understand the genetic diversity of M. oryzae from eastern India that would guide in the development of more effective control strategies and improving rice blast resistance through development of new resistant varieties through marker-assisted breeding.

Genetska varijabilnost unutar i između generativnih potomstava divlje ruže (Rosa sp.)

Dog rose (Rosa canina L.) is a cosmopolitan plant species that due to its nutritional, medical and cosmetic values deserves more attention. Dog roses are cultivated in Croatia on very small scale and most of the rose hip used in the domestic processing industry is imported. In this study, three dog rose F1 progeny populations developed from selected plants collected in Korcula, Malesnica and Prigorje Brdovecko, originating from two climatic regions (P1 - Mediterranean and P2 and P3 - continental) have been studied as a part of a field experiment for two growing seasons, in order to assess the level of genetic variability for pomological and agronomic traits. In addition, genetic variation within and among examined three dog rose populations has been assessed using 6 polymorphic SSR markers. The highest variability, as well as the most desirable agronomic traits were found within the P3 population, while the lowest ones were present in the P1 population. Most desirable properties and highest values for commercially important traits were detected in the genotype RC29 (P3). Molecular data was sufficient to separate three studied populations but not all individuals within the populations. Through selection and hybridization of perspective genotypes of dog roses from Croatia, it would be possible to create clonally propagated varieties suitable for commercial cultivation.

Parental evaluation and polymorphism survey of drought contrasting donor and recurrent parents in rice (Oryza sativa. L) using microsatellite markers

Rice is an important food crop which is considered as lifeline for more than half of the world's population. Rice requires larger amount of water throughout its life cycle than other crops. Parental lines of Kasturi and Chaw Khao were evaluated and exhibited variation for panicle length, plant height and plot yield in stress condition. Association analysis also shown plant height and plot yield was positively associated with plot yield. In this study parental polymorphism survey was conducted between Kasturi and Chaw Khao using 721 microsatellite (SSR) markers spanning over the entire 12 chromosome and resulted in 95 polymorphic SSR markers. The overall polymorphism survey for all 12 chromosomes was found to be 13.17%. The highest and lowest polymorphism was noticed in chromosome 5 (17.02 %) and chromosome 10 (5.36 %), respectively. This investigation will be helpful to genotype mapping population, construction of linkage map and grain yield QTL identification for drought stress.

Genome-wide association mapping of stress-tolerance traits in cotton

Environmental stresses severely impair cotton production worldwide. To identify the genetic basis of, and molecular markers associated with, environmental stresses such as salt, cold and Verticillium wilt, association mapping of salt-, cold-, and disease-tolerance traits was performed in a population of 503 upland cotton accessions using 179 polymorphic SSR markers and 11,975 array-derived SNP markers. Salt and cold tolerance was evaluated via the relative germination rate (RGR) of the seeds under seven and four environments, respectively. The disease index of Verticillium wilt was investigated for two years in the field in Xinjiang. These three traits showed large variation across environments. A genome-wide association study revealed that 31, 19, and 15 SSR markers were associated with RGR-Salt (the relative germination rates of seeds under salt stress), RGR-Cold (the relative germination rates of seeds under cold stress), and DIV (the disease index of Verticillium wilt), respectively. Six SNPs in seven environments and two SNPs in BLUP (best linear unbiased prediction) results were associated with RGR-Salt, and the phenotypic variance explained ranged from 3.96 to 5.00%. Two SNPs (i02237Gh, i02243Gh) on D01 were concluded to be stable genetic loci associated with RGR-Salt. A total of 223 genes were found in a candidate gene interval (D01, 37771–1942912). Of these four genes, GhPIP3A, GhSAG29, GhTZF4, and GhTZF4a, showed expression changes in sensitive and tolerant genotype accessions under salt stress, and were assigned as candidate genes associated with RGR-Salt. This study revealed the genetic basis of stress resistance in upland cotton and will facilitate stress-resistance breeding in cotton.

De Novo Transcriptomic Analysis and Development of EST–SSRs for Styrax japonicus

Styrax japonicus sieb. et Zucc. is widely distributed in China with ornamental and medicinal values. However, the transcriptome of S. japonicus has not yet been reported. In this study, we carried out the first transcriptome analysis of S. japonicus and developed a set of expressed sequence tag–simple sequence repeats (EST–SSRs). We obtained 338,570,222 clean reads in total, of which the mean GC content was 41.58%. In total, 136,071 unigenes were obtained having an average length of 611 bp and 71,226 unigenes were favorably annotated in the database. In total, we identified 55,977 potential EST–SSRs from 38,611 unigenes, of which there was 1 SSR per 6.73 kb. The di-nucleotide repeats (40.40%) were the most identified SSRs. One set of 60 primer pairs was randomly selected, and the amplified products in S. japonicus were validated; 28 primer pairs successfully produced clear amplicons. A total of 21 (35%) polymorphic genic SSR markers were identified between two populations. In total, 15 alleles were detected and the average number was 6. The average of observed heterozygosity and expected heterozygosity was 0.614 and 0.552, respectively. The polymorphism information content (PIC) value fluctuated between 0.074 and 0.855, with a mean value of 0.504, which was also the middle level. This study provides useful information for diversity studies and resource assessments of S. japonicus.

Breeding lines of the Indian mega-rice variety, MTU 1010, possessing protein kinase OsPSTOL (Pup1), show better root system architecture and higher yield in soils with low phosphorus

MTU 1010 is a high-yielding mega-variety of rice grown extensively in India. However, it does not perform well in soils with low phosphorus (P) levels. With an objective to improve MTU 1010 for tolerance to low soil P, we have transferred Pup1, a major quantitative trait locus (QTL) associated with tolerance from another mega-variety, Swarna, through marker-assisted backcross breeding (MABB). Foreground selection of the F1 and backcross plants was performed with the co-dominant, closely linked CAPS marker, K20-2, while two flanking markers RM28011 and RM28157 were utilized for recombinant selection. At each backcross generation, positive plants were also analyzed with a set of 85 parental polymorphic SSR markers to identify the QTL-positive plants possessing maximum introgression of MTU 1010 genome. At BC2F1, the best backcross plant was selfed to generate BC2F2s. Among them, the plants homozygous for Pup1 (n = 22) were reconfirmed using the functional marker for Pup1, viz., K46-1, and they were advanced through pedigree method of selection until BC2F6 generation. A total of five elite BC2F6 lines, possessing Pup1 and phenotypically similar to MTU 1010, were screened in the low soil P plot and normal plot (with optimum soil P levels) during wet season, 2016. All the selected lines showed better performance under low P soil with more number of productive tillers, better root system architecture, and significantly higher yield (> 390%) as compared to MTU 1010. Further, under normal soil, the lines were observed to be similar to or better than MTU 1010 for most of the agro-morphological traits and yield. This study represents the successful application of marker-assisted selection for improvement of tolerance to low soil P in a high-yielding Indian rice variety.

Genetic diversity and genetic variation in morpho-physiological traits to improve heat tolerance in Spring barley.

Heat stress is one of the abiotic stresses that limit the production and productivity of barley. Understanding the genetic variation, changes in physiological processes and level of genetic diversity existing among genotypes are needed to produce new cultivars not only having a high tolerance to heat stress, but also displaying high yield. To address this challenge, a set of 60 highly homozygous, diverse barley genotypes were evaluated under normal and heat stress conditions in two seasons of 2014/2015 and 2015/2016. Seedling vigor (SV) as a morphological trait was visually scored under normal conditions. Plant height (Ph), days to flowering (DOF), 1000-kernel weight (TKW), grain yield per spike (GYPS), yield per plot (YPP) and biological yield (BY) were measured. Moreover, proline content (ProC), soluble carbohydrate content (SCC), starch content, soluble protein (SP), and amino acid (AA) content as physiological parameters were analyzed from the grains. High genetic variation was observed among genotypes for all traits scored in this study. All traits had high broad-sense heritability estimates ranging from 0.59 (SV) to 0.97 (TKW) for yield traits. Seedling vigor was significantly correlated with all yield traits under both conditions. Among all physiological traits, the increase in ProC and reduction in starch content due to heat stress had significant correlations with the reduction due to heat stress in YPP, GYPS, TKW, and BY. Furthermore, the genetic diversity based on genetic distance (GD) among genotypes was investigated using 206 highly polymorphic SSR marker alleles. The GD ranged from 0.70 to 0.98 indicating that these genotypes are highly and genetically dissimilar. The combination of analyses using molecular markers, genetic variation in yield traits, and changes in physiological traits provided useful information in identifying the tolerant genotypes which can be used to improve heat tolerance in barley through breeding.

Paternity analysis using SSR markers reveals that the anthocyanin-rich tea cultivar ‘Ziyan’ is self-compatible

Most tea plants (Camellia sinensis) are self-incompatible. Self-incompatibility is critical for preventing inbreeding depression and maintaining genetic diversity, but it can hinder breeding and genetic studies. Originally, the propose of this study was to identify fathers of 16 elite F1 individuals derived from open-pollinated seeds of an anthocyanin-rich tea cultivar ‘Ziyan’. In order to achieve this, 41 candidate parents and 16 F1 individuals were genotyped with 15 polymorphic SSR markers. Using exclusion and likelihood methods, fathers of 12 individuals were identified at 95% level of confidence. Surprisingly, the results showed that eight individuals were from self-crossing of ‘Ziyan’. Additional 36 SSR markers were used to further confirm selfing and the results were consistent, demonstrating that ‘Ziyan’ is self-compatible. As expected, the degrees of heterozygosity of the eight selfing individuals were significantly lower than ‘Ziyan’ and other tea accessions. This is the first report of a clonal cultivar of C. sinensis being self-compatible under open-pollination conditions and verified using DNA-based markers. Therefore, ‘Ziyan’ and its selfing offspring will be valuable materials for both genetic studies and breeding in C. sinensis.

Marker-assisted pyramiding of two major, broad-spectrum bacterial blight resistance genes, Xa21 and Xa33 into an elite maintainer line of rice, DRR17B.

Bacterial blight (BB) disease reduces the yield of rice varieties and hybrids considerably in many tropical rice growing countries like India. The present study highlights the development of durable BB resistance into the background of an elite maintainer of rice, DRR17B, by incorporating two major dominant genes, Xa21 and Xa33 through marker-assisted backcross breeding (MABB). Through two sets of backcrosses, the two BB resistance genes were transferred separately to DRR17B. In this process, at each stage of backcrossing, foreground selection was carried out for the target resistance genes and for non-fertility restorer alleles concerning the major fertility restorer genes Rf3 and Rf4, using gene-specific PCR-based markers, while background selection was done using a set of 61 and 64 parental polymorphic SSR markers respectively. Backcross derived lines possessing either Xa21 or Xa33 along with maximum genome recovery of DRR17B were identified at BC3F1 generation and selfed to develop BC3F2s. Plants harboring Xa21 or Xa33 in homozygous condition were identified among BC3F2s and were intercrossed with each other to combine both the genes. The intercross F1 plants (ICF1) were selfed and the intercross F2(ICF2) plants possessing both Xa21 and Xa33 in homozygous condition were identified with the help of markers. They were then advanced further by selfing until ICF4 generation. Selected ICF4 lines were evaluated for their resistance against BB with eight virulent isolates and for key agro-morphological traits. Six promising two-gene pyramiding lines of DRR17B with high level of BB resistance and agro-morphological attributes similar or superior to DRR17B with complete maintenance ability have been identified. These lines with elevated level of durable resistance may be handy tool for BB resistance breeding.

Improved versions of rice maintainer line, APMS 6B, possessing two resistance genes, Xa21 and Xa38, exhibit high level of resistance to bacterial blight disease

APMS 6B is the stable maintainer of the CMS line APMS 6A, which is the female parent of the popular Indian rice hybrid DRRH 3. APMS 6B has good combining ability and plant stature but is highly susceptible to bacterial blight (BB) disease. In order to improve the BB resistance of APMS 6B, we pyramided two major, dominant BB resistance genes, Xa21 and Xa38, through marker-assisted backcross breeding (MABB). Improved Samba Mahsuri (ISM) was used as the donor for Xa21 while PR 114 (Xa38) served as the donor for Xa38. Individual crosses [APMS 6B/ISM and APMS 6B/PR 114 (Xa38)] were performed, and true F1 plants were then backcrossed with APMS 6B and the MABB process was continued till BC3. A single positive BC3F1 plant identified from both the crosses with maximum genotypic and phenotypic similarity with APMS 6B was selfed to generate BC3F2s. At BC3F2 generation, plants homozygous for either Xa21 or Xa38 were identified and further confirmed for the absence of two major fertility restorer genes, Rf3 and Rf4. A single such homozygous BC3F2 plant, each from both the crosses, was then inter-mated to generate ICF1s (inter-cross F1s). Selected ICF1 plants possessing both the BB resistance genes were selfed to generate ICF2s. A total of 42 ICF2 plants homozygous for both Xa21 and Xa38 were identified and screened with parental polymorphic SSR markers to identify the best F2 plants having the maximum recurrent parent genome recovery. Twelve best ICF2 plants were advanced up to ICF5. The ICF5 lines displayed very high level of BB resistance and were similar to APMS 6B in terms of agro-morphological characters. Further, most of these lines also showed complete maintenance ability and such lines are being advanced for conversion to WA-CMS lines.

Genetic analysis and marker assisted backcrossing for transfer of mosaic virus resistance in cowpea [Vigna unguiculata (L.) Walp.

Cowpea mosaic virus (CpMV) is known to cause yield loss of as much as 80-100 per cent. Among different approaches available in the management of this disease development and release of resistant varieties is the most viable option for farmers. Hence an investigation was carried out to study the inheritance of resistance to cowpea mosaic virus (CpMV) and to identify DNA markers linked to genomic regions conferring resistance to CpMV. The resistance was found to be governed by single recessive gene in the 191 F2:3 progenies derived from the cross C-152 (susceptible) × V-57817 (resistant). Single marker analysis with 191 F2 individuals and 106 polymorphic SSR markers indicated that two markers, MA15 and MA80 were linked to CpMV resistance as evidenced by significant mean sum of squares due to between marker classes and putative linkage of these markers for resistance to CpMV was confirmed by bulked segregant analysis. Through linkage map constructed utilizing 91 polymorphic SSR markers it was possible for us to show that the two markers MA15 and MA80 were included in the linkage group 6 and the marker gene was likely to be present between these markers. Marker assisted backcrossing was practiced to transfer resistance gene in to an agronomically superior variety C-152.

Development and characterization of novel set of polymorphic SSR markers for Dill (Anethum graveolens L.)

Dill ( Anethum graveolens L.), belonging to the Apiaceae family, is considered one of the most important and economically important seed spice grown and cultivated all over India. But, due to minimal genetic and genomic resources, most of the past studies were based on physiological and biochemical analysis rather than molecular breeding. In the present study an attempt was made to develop and design in-silico and computationally derived highly informative microsatellite markers. Current investigation was the first attempt to develop and design microsatellite markers from Dill EST and genomic sequences. A total of 1556 SSRs were found with a density of 1SSR/0.38 kb in 589.390 kb sequence searched. Of the total, 1265, 81.30% were perfect and 291, 18.70% were identified as compound SSRs in nature. Mononucleotides (1089, 69.98%) were most frequent followed by trinucleotides (254, 16.32%), dinucleotides (183, 11.76%), tetranucleotides (22, 1.41%), hexanucleotides (6, 0.38%) and the least frequent were pentanucleotides (2, 0.13%).

Development of novel EST-SSR markers for Rhododendron longipedicellatum (Ericaceae) and cross-amplification in two congeners.

Premise of the StudyTo investigate the genetic background and population characteristics of Rhododendron longipedicellatum (Ericaceae), a newly discovered and critically endangered species, expressed sequence tag–simple sequence repeat markers were developed, and transferability was tested in two congeners, R. molle and R. simsii.Methods and ResultsBased on the transcriptome sequences of R. longipedicellatum, 102 primer sets were designed; 48 primer sets were successfully amplified, with 15 showing polymorphisms in 150 individuals from five extant populations of R. longipedicellatum. The number of alleles per locus ranged from four to 18, and the levels of observed and expected heterozygosity for the 15 loci varied from 0.255 to 0.913 and from 0.306 to 0.851, respectively. All 15 loci were found to amplify in R. molle and R. simsii.ConclusionsThese polymorphic SSR markers can be used in conservation genetic and phylogeographic studies to elucidate the rarity and origin of R. longipedicellatum.

Marker Assisted Selection for Sheath Blight, Blast and Bacterial Blight Resistance in Two Popular Rice Varieties

Major biotic stresses that affects rice production are sheath blight (ShB) caused by Rhizoctonia solani Kuhn, blast disease caused by fungal pathogen Magnaporthe oryzae and bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae. To reduce the yield loss pyramiding of resistance (R) genes in an elite rice variety is the most effective method. Two high yielding rice varieties namely, ASD16 and ADT43 are extensively grown in South India. These varieties are susceptible to blast, bacterial blight and sheath blight diseases. Hence this study aims to improve these disease susceptible varieties to achieve higher yield in disease prone areas. Improved ASD16 (CB14004) harbouring BB and blast and improved ADT43 (CB14002) harbouring BB resistance genes were used in the present marker assisted backcross breeding program to introgress three sheath blight resistant QTLs (qSBR 11-1, qSBR 11-2 and qSBR 7-1). In every backcross generation, PCR-based markers (RM224, RM209 and RM336), linked with sheath blight resistance QTLs, bacterial blight resistance genes (xa5, xa13 and Xa21) and blast resistance genes (Pi54) were utilized for foreground selection. In addition, a set of forty eight (CB14002 x tetep) and forty one (CB14002) parental polymorphic SSR markers were used for background selection and backcrossing was carried out until BC3 generation. Background selection studies using polymorphic markers revealed that, an average recovery of 91.15% and the maximum of 92.68% recurrent parent genome in CB14004 x Tetep cross population. In CB14002 x Tetep an average of 90.20% and maximum of 92.70% has been recovered. These plants will be selfed to generate large number of segregating progenies to identify agronomically superior with good grain quality traits and multiple disease resistance lines in rice..

Incorporation of the novel bacterial blight resistance gene Xa38 into the genetic background of elite rice variety Improved Samba Mahsuri.

Bacterial blight (BB) in rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major global production constraint, particularly in irrigated and rain-fed lowland areas. Improved Samba Mahsuri (ISM) is an elite, high-yielding, fine-grain type, BB-resistant rice variety possessing three BB-resistant genes (Xa21, xa13 and xa5) and is highly popular in the southern parts of India. As the BB pathogen is highly dynamic and the evolution of pathogen virulence against the deployed resistance genes is common, we added a novel BB-resistant gene, Xa38, into ISM through marker-assisted backcross breeding (MABB) to increase the spectrum and durability of BB resistance. The breeding line PR 114 (Xa38) was used as the donor for Xa38, whereas ISM was used as the recurrent parent. Foreground selection was conducted using PCR-based gene-specific markers for the target genes, whereas background selection was conducted using a set of polymorphic SSR markers between the parents and backcrossing that continued until the third generation. Eighteen homozygous BC3F2 plants possessing all four BB-resistant genes in the homozygous state and with a recurrent parent genome (RPG) recovery of more than 92% were identified and advanced to the BC3F6 generation. These 18 backcross-derived lines (BDLs) exhibited very high level of resistance against multiple Xoo strains and displayed agro-morphological traits, grain qualities and yield levels similar to or better than those of the recurrent parent ISM.

Improvement of blast resistance of the popular high-yielding, medium slender-grain type, bacterial blight resistant rice variety, Improved Samba Mahsuri by marker-assisted breeding

Improved Samba Mahsuri (ISM) is a popular, high-yielding, bacterial blight resistant rice variety possessing medium-slender grain type. As ISM is highly susceptible to blast disease of rice, through the present study we have transferred two major blast resistance genes, Pi2 and Pi54 into the elite variety by marker-assisted backcross breeding. The two blast resistance genes were transferred to ISM through sets of backcrosses. In every backcross generation, PCR-based markers, specific for the blast resistance genes (Pi2 and Pi54) and bacterial blight resistance genes (Xa21, xa13 and xa5) were utilized for foreground selection, while a set of 144 parental polymorphic SSR markers were used for background selection and backcrossing was carried out until BC2 generation. A solitary BC2F1 plant possessing Pi2 or Pi54 along with Xa21, xa13 and xa5 and > 90% recovery of ISM genome was selected from the two sets of backcrosses were crossed and the intercross F1s (ICF1s) thus obtained were selfed to generate ICF2s. Homozygous ICF2 plants carrying all the five resistance genes were identified through markers and advanced through selfing till ICF5 generation by adopting pedigree method of selection. Three best lines at ICF5, possessing excellent resistance against bacterial blight and blast and closely resembling or superior to ISM in terms of grain quality: yield and agro-morphological traits have been identified and advanced for multi-location trials.

Genetic Evaluation of Natural Populations of the Endangered Conifer Thuja koraiensis Using Microsatellite Markers by Restriction-Associated DNA Sequencing.

Thuja koraiensis Nakai is an endangered conifer of high economic and ecological value in Jilin Province, China. However, studies on its population structure and conservation genetics have been limited by the lack of genomic data. Here, 37,761 microsatellites (simple sequence repeat, SSR) were detected based on 875,792 de novo-assembled contigs using a restriction-associated DNA (RAD) approach. Among these SSRs, 300 were randomly selected to test for polymorphisms and 96 obtained loci were able to amplify a fragment of expected size. Twelve polymorphic SSR markers were developed to analyze the genetic diversity and population structure of three natural populations. High genetic diversity (mean NA = 5.481, HE = 0.548) and moderate population differentiation (pairwise Fst = 0.048–0.078, Nm = 2.940–4.958) were found in this species. Molecular variance analysis suggested that most of the variation (83%) existed within populations. Combining the results of STRUCTURE, principal coordinate, and neighbor-joining analysis, the 232 individuals were divided into three genetic clusters that generally correlated with their geographical distributions. Finally, appropriate conservation strategies were proposed to protect this species. This study provides genetic information for the natural resource conservation and utilization of T. koraiensis and will facilitate further studies of the evolution and phylogeography of the species.

Heterotic Group Classification of 63 Inbred Lines and Hybrid Purity Identification by Using SSR Markers in Winter Cabbage (Brassica Oleracea L. var. capitata)

Winter cabbage is an important crop cultivated through winter in the region near the Yangtze River, enabling the supply of fresh cabbage there at that time of year. However, a problem has emerged regarding the newly developed parents of winter cabbage, which is completely different from spring and autumn cabbage, namely, how to combine these parents to breed an elite hybrid. To classify the heterotic groups and improve the efficiency of parent selection in winter cabbage breeding, 20 polymorphic SSR markers were selected to screen 63 winter cabbage inbred lines. Seventeen pairs among the 20 SSR markers amplified polymorphic bands. These primers amplified two to six bands, with an average of 2.8 bands per primer, and a total of 47 polymorphic bands were generated in the 63 inbred lines. These lines included flat-headed morphotype and round-headed morphotype, thus they were separately classified into heterotic groups based on the SSR markers. The flat-headed morphotype contained 21 inbred lines and was classified into three heterotic groups, named Hanchun 4, Jiali, and Dongsheng, in accordance with the representative germplasm contained in each group. The round-headed morphotype contained 42 inbred lines and was classified into five heterotic groups, named Parte, Bejo1039, YK-143, SCA002, and Golden B90. Meanwhile, parent analysis of 20 developed elite combinations showed that their parents were all distributed in different heterotic groups, indicating that the group classification was reasonable, which can provide a basis for further parent selection in winter cabbage breeding. Furthermore, polymorphic SSR primers were successfully used to identify the hybrid purity of three elite varieties.