| Walnut(Juglans regia, 2n=32) is one of the most important woody grain and oil strategic tree species in the world that has very high nutritional value of kernel and ecological value, approximately 606 Mb per 1C genome. Walnut anthracnose caused by Colletotrichum gloeosporioides is one of the most serious walnut diseases, which leads to sharp reduction of quality and yield. It is important link of molecular marker-assisted breeding that constructs high-density genetic linkage map of walnut by molecular markers and locating the Quantitative trait loci(QTL) of anthracnose resistance trait. In this research, a high density genetic linkage map of walnut was constructed with Specific length amplified fragment sequencing(SLAF-seq) in walnut cultivar ?Yuan Lin?( female parent), cultivar ?Qing Lin?(male parent) and 84 F1 progeny derived from a cross of walnut cultivar ?Yuan Lin? and cultivar ?Qing Lin?. The QTL of anthracnose resistance were analyzed. The main results were as follows:1. Analysis of SLAF-seq data and SLAF markers: A total of 161.64 M pair-end reads were generated, a number of 153,820 SLAF markers were obtained, whose average depth was 68.06 in the female parent, 56.21 in the male parent and 7.91 in the progeny. Among these markers, there were 49,174 polymorphism markers with polymorphism rate of 31.97%, 38,664 markers were genotyped successfully and were classified into eight segregation types. After filtering low quality SLAF markers, segregation distortion markers and other markers which were not suitable for mapping, 13,635 polymorphism markers were obtained and were sorted to five segregation types(ef×eg: 962, hk×hk: 1,046, lm×ll: 3,501, nn×np: 8,123, ab×cd: 3).2. High-density genetic linkage map: After 13,635 SLAF markers for linkage analysis, eventually, mapping markers contained 2,577 SLAF markers, and 761 of them showed significant segregation distortion, accounted for 29.53% of mapping markers. 2,395 of these fell into 16 linkage groups(LGs) for the female map, 448 markers for the male map, and 2,577 markers for the integrated map. Taking into account the size of all LGs, marker coverage amounted to 2,664.36 c M for the female map, 1,305.58 c M for the male map, and 2,457.82 c M for the integrated map. The average intervals between two adjacent mapped markers were 1.11 c M, 2.91 c M and 0.95 c M for female maps, male maps, and integrated maps. Every linkage group of the female map was consisted with 1~242 SLAF markers, and the length of linkage group was between 0.00~274.38 c M; Every linkage group of the male map was consisted with 1~80 SLAF markers, and the length of linkage group was between 0.00~174.78 c M; Every linkage group of the integrated map was consisted with 80~243 SLAF markers, and the length of linkage group was between 78.12~190.04 c M.3. Among mapping markers, there were three types of markers including ?SNP_only?, ?In Del_only? and ?SNP&In Del?. ?SNP_only? was the predominant marker type accounting for 89.25% of mapping markers. A number of 5,043 Single nucleotide polymorphisms(SNP) loci were detected among 2,577 markers, which mean 2 SNP loci per SLAF marker. In total, most of the SNPs were transition type SNPs containing R(G/A) and Y(T/C) types accounting for 33.61% and 34.62% of all SNPs, respectively.4. The QTL analysis of walnut anthracnose resistance: According to constructed integrated maps, we took interval mapping(Logarithm of odds, LOD>3.0) to detect our quantitative trait. A total of one QTL was detected for anthracnose resistance, and ranged from 165.51 to 176.33 c M on LG14. Ten markers above the threshold value were considered to be linked markers to anthracnose resistance trait, which pecent phenotypic variance explained ranged from 16.2% to 19.9%. |
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