A bacterial artificial chromosome(BAC) library was constructed for Gossypium hirsutum acc.TM-1,a genetic and genomic standard line for Upland cotton.The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb.About 96.0% of the clones have inserts over 100 kb.Therefore,this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb.Clones were stored in 384 384-well plates and arrayed into multiplex pools for rapid and reliable library screening.BAC screening was carried out by four-round polymerase chain reactions using 23 simple sequence repeats(SSR) markers,three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library.Correspondingly,in total 92 positive BAC clones were identified with an average four positive clones per SSR marker,ranging from one to eight hits.Additionally,since these SSR markers have been localized to chromosome 12(A12) and 26(D12) according to the genetic map,these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes,sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.
Sub 16 is a substitution line with G. hirsutum cv. TM-1 genetic background except that the 16th chro-mosome (Chr. 16) is replaced by the corresponding homozygous chromosome of G. barbadense cv. 3-79, and T586 is a G. hirsutum multiple gene marker line with 8 dominant mutation genes. The R1 gene for anthocyanin pigmentation was tagged in Chr. 16 in T586. The objective of this research was to screen SSR markers tightly linked with R1 by using the F2 segregating population containing 1259 plants derived from the cross of Sub 16 and T586 and the backbone genetic linkage map from G. hir-sutum×G. barbadense BC1 newly updated by our laboratory. Genetic analysis suggested that the seg-regation ratio of red plants in the F2 population fit Mendelian 1:2:1 inheritance, confirming that the red plant trait was controlled by an incomplete dominance gene. Preliminary mapping of R1 was conducted using 237 randomLy selected F2 individuals and JoinMap v3.0 software. Then, a fine map of R1 was constructed using the F2 segregating population containing 1259 plants, and R1 was located between NAU4956 and NAU6752, with only 0.49 cM to the nearest maker loci (NAU6752). These results pro-vided a foundation for map-based cloning of R1 and further development of cotton cultivars with red fibers by transgenic technology.
ZHAO Liang CAI CaiPing ZHANG TianZhen GUO WangZhen
Simple sequence repeats(SSRs) have been widely applied as molecular markers in genetic studies.However,the number of expressed sequence tags(ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species.In this study,EST-SSR distribution from G.barbadense was characterized and new G.barbadense-derived EST-SSR markers were determined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G.barbadense.By mining 9697 non-redundant ESTs,a total of 638 SSR loci derived from 595 ESTs were observed.In G.barbadense,the frequency of ESTs containing SSRs was 6.13%,with an average of 1 SSR in every 10.4 kb of EST sequence.Furthermore,trinucleotide was found to be the most abundant repeat type among 2-6-nucleotide repeat types.It accounted for 26.6% of the total,followed by the hexanucleotide(26.0%) and pentanucleotide repeats(25.9%).Among all the repeat motifs,(AAG)n accounted for the highest proportion.EST-SSR primer pairs were developed using the Primer3 program,and the redundant primers were removed using the virtual PCR approach.As a result,380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G.hirsutum 'TM-1' and G.barbadense 'Hai7124' for constructing linkage groups in cultivated allotetraploid cotton.Out of these,98(25.8%) primer pairs detected polymorphisms.Finally,95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map;of these,42 were mapped into the A subgenome and 53 into the D subgenome.The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species.
LU YuanDa CAI CaiPing WANG Lei LIN ShaoYan ZHAO Liang TIAN LiangLiang LU JunHong ZHANG TianZhen GUO WangZhen
Verticillium wilt(caused by the pathogen Verticillium dahliae) is of high concern for cotton producers and consumers. The major strategy for controlling this disease is the development of resistant cotton(Gossypium spp.) cultivars. We used interspecific chromosome segment introgression lines(CSILs) to identify quantitative trait loci(QTL) associated with resistance to Verticillium wilt in cotton grown in greenhouse and inoculated with three defoliating V. dahliae isolates. A total of 42 QTL, including 23 with resistance-increasing and 19 with resistancedecreasing, influenced host resistance against the three isolates. These QTL were identified and mapped on 18 chromosomes(chromosomes A1, A3, A4, A5, A7, A8, A9, A12, A13, D1, D2,D3, D4, D5, D7, D8, D11, and D12), with LOD values ranging from 3.00 to 9.29. Among the positive QTL with resistance-increasing effect, 21 conferred resistance to only one V. dahliae isolate, suggesting that resistance to V. dahliae conferred by most QTL is pathogen isolate-specific. The At subgenome of cotton had greater effect on resistance to Verticillium wilt than the Dt subgenome. We conclude that pyramiding different resistant QTL could be used to breed cotton cultivars with broad-spectrum resistance to Verticillium wilt.
Chromosome segment substitution lines (CSSL) consist of a battery of nearisogenic lines that have been developed and cover the entire genome of some crops. With the exception of one homozygous chromosome segment transferred from a donor parent, the remaining genome of each CSSL line is the same as the recipient parent. It is an ideal material for genome research and particularly QTL mapping. In the present study, we first developed one set of CSSL lines using G. hirsutum acc. TM-1 (the genetic standard), as the recipient parent and G. barbadense cv. Hai7124 as the donor parent using molecular assisted-selection in BC5S1-3 generations. The CSSL consisted of 330 different lines, in which 1-4 different lines had the same or overlapping substituted segments. The genetic length of the substituted segments covered 5271.9 cM with an average segment distance of 10.9 cM, 1.5 times the total genetic length of Upland cotton (3514.6 cM). The substituted segments of each line varied in length, ranging from 3.5 cM for the shortest segment to 23.2 cM in the longest segment. Our CSSL have not yet to cover the entire tetraploid cotton genome, due to the absence of some donor parent interval segments.
WANG Peng DING YeZhang LU QiongXian GUOWangZhen ZHANG TianZhen
