Genetic Diversity And Molecular Markers Of Yellow Seed And Erucic Acid In Brassica Juncea

Oilseed rape, one of the four top oil crops in the world, is the main source of plant edible oil. Brassica juncea, of rich genetic resources, originated in China with many excellent characteristics, such as drought tolerance, high temperature resistance, tolerance to poor soil tolerance, shattering resistance, yellow seed, early maturity, late sowing tolerance, disease resistance and heavy metal tolerance etc. Mustard is not only suitable for planting in drought area but also the valuable resources for the genetic improvement of Brassica napus. The yellow mustard in Northern Shaanxi, distributed in the Loess Plateau and its vicinity, is a unique ecological type. The yellow seededness and special fatty acid composition are both the breeding target traits in rapeseed breeding. In this paper the genetic diversity of B. juncea in west part of China, the yellow seed trait of yellow mustard in Northern Shaanxi and its fatty acid composition were analyzed by classic genetics and molecular biological methods.The results as follows:1. The genetic diversity of B. juncea in the west of ChinaOf 101 accessions of B. juncea from different ecological zones in the west of China and seven controls included 2 mustard lines from abroad, 4 Brassica rapa lines and 1 Eruca sativa Mill line were been used to analyze the genetic diversity by means of SRAP,AFLP and SSR techniques. The main results were as follows:1.1 The 101 accessions of B. juncea and 2 mustard lines from abroad were divided into 5 groups, those were group A from Yunnan-Guizhou and Southern Shaanxi, group B from Guanzhong of Shaanxi, group C and group D from Xinjiang, and group E from western China. Groups A and B were winter type, groups C, D, and E were spring type. The geological and biological conditions were the main factors to influence the classification of B.juncea.1.2 The genetic difference in group A was the largest, and higher than that in group B. The accessions from Shaanxi and Xinjiang were distributed into 3 groups respectively, and showed abundant genetic diversity. The genetic diversities in winter type of B. juncea were higher than those in spring type in China.1.3 Group E including the most spring accessions, was divided into 3 sub-groups. The accessions in the sub-group I from Tibet, with the genetic similarity coefficient higher than 0.83, belonged to an independent genetic system with narrow genetic background. The accessions in the sub-group II from Northern Shaanxi and its vicinity showed higher genetic diversity and belonged to another independent genetic population. In the sub-group III, two accessions from Australia were similar to the spring type in China2. The genetic analysis and mapping on yellow seed trait of yellow mustard in Northern ShaanxiTwo brown seeded Brasscia juncea cultivars (Wugong mustard and Chang-an mustard) were crossed with Wuqi yellow mustard lines (yellow seed trait),respectively. The genetic mechanism of yellow seed trait was then analyzed with classic genetics method. AFLP combined with BSA technique was exploited to detect the molecular markers closely linked to the yellow seed trait using an F2 population (Wuqi yellow mustard×Wugong brown mustard).The main results were as follows:2.1 The seed colour examination of F0 and F1 of two reciprocal crosses indicated that the seed colour was governed by the maternal embryo gene, in which brown seed trait was dominant over yellow seed. The seed colour examination of F2, BC1 (yellow seed cultivar as male parent) and BC2 (brown seed cultivar as male parent) showed that the segregation ratio of brown seed to yellow seed fitted to 3:1, 1:1 and 1:0, respectively, which meant that the seed colour of the yellow mustard was controlled by one pair of major genes.2.2 The correlation analysis between several quality characters and the seed colour of F2 population derived from yellow×brown showed there existed strong correlation between oil content and seed colour but no distinct correlation existed between the mean value of fatty acid composition and seed colour.2.3 512 AFLP primer combinations (256 P0+3/MC+2 and 256 P0+3/MG+2) were screened between two parents and two bulks. Further testing the corresponding individuals composing the two bulks 16 AFLP markers tightly linked to the seed colour trait.2.4 Except marker P13MG04 which did not have bands the rest 15 markers were distributed in one linkage group by linkage analysis 16 AFLP markers in 346 individuals of F2 population. These 15 AFLPs were located on both sides of the gene controlling seed colour and the linkage map covered 38.9cM of the whole genome, where the average distance between markers was 2.59cM. P11MG15 was co-segregated with the target gene; the closest markers on the both sides of the seed colour gene were P03MC08, P16MC02 and P11MG01, the distance of which were 0.3cM, 0.3cM, and 0.7cM, respectively.2.5 16 AFLPs were been converted to SCAR markers successfully and 6 SCARs ( SG6-4F/4R, SG2-1F/1R,SG2-2F/2R, SC11-1F/1R,SC11-2F/ 2R and SC8-2F/2R) were been obtained (which were derived from P11MG01, P15MG15, P09MC12 and P16MC02 ) respectively. Screened F2 population with the 6 SCARs the results were consistent to those of AFLP. SG6-4F/4R and SC8-2F/2R on the opposite side of the target gene were the closest markers and the genetic distance were 0.7cM and 0.3cM, respectively. SCARs SG2-1F/1R(SG2-2F/2R) and SC11-1F/1R(SC11-2F/2R) were relatively far from the seed colour gene and the genetic distance were 20.9cM and 28.3cM, respectively.3. The heredity of erucic acid content (EAC) , the cloning and sequence alignments of FAE1 of yellow mustard in Northern ShaanxiIn order to elucidate the genetic mechanism of erucic acid in yellow mustard of Northern Shaanxi, classic genetics method was exploited to analyse the progenies of Chang-an mustard (EAC 45%)×Wuqi yellow mustard (EAC 25.6%) and 4923 (EAC 0.86%)×Wuqi yellow mustard. To better understand the composition feature of FAE1 in the yellow mustard and its low erucic acid mutant (1278-3), and SNPs of FAE1 relate to the EAC variation, the FAE1 genes were isolated, sequenced and compared to different types and different sources of mustards and related species of Brassica genus using homological sequence method. The main results were as follows:3.1 The EAC of the yellow mustard was governed by two pairs of additive embryo genes, one of which was dominant homozygous (E1E1 or E2E2) controlling 24% of EAC and the other one was recessive homozygous (e2e2 or e1e1) controlling less than 4% of EAC. High EAC (E1, E2) was partially dominant to low EAC.3.2 The FAE1 sequence from our material was 1353bps and located from 110bp to 1463bp in the published FAE1 sequence. ORF analysis showed that FAE1 gene contained only one open reading frame without intron, which was consistent with the previous result. Sequence alignments indicated that the homology ratio of our sequence was over 88.4% comparing with the published Arabidopis thaliana FAE1 (U29142) sequence, and over 95% with the FAE1 sequence from species of Brassica genus. Furthermore, the homology ratio was more than 99% comparing with the published Brassica juncea FAE1 sequences: AF558197(FAE1.1) and AF558198(FAE1.2) ₃.3 FAE1 genes of B. juncea were divided into BJFAE1.1 and BJFAE1.2 two types. There were 62 base differences between BJFAE1.1 and BJFAE1.2, of which 11 differences were missense mutations and the rest were synonymous mutations. There was loss of a HindIII site in BJFAE1.1 at position 1144 and at position 1415 in BJFAE1.2. The base difference at position 591 and 1265 in BJFAE1.1 and at position 237 in BJFAE1.2 were related to genomes A and B.Homology was very high among the two FAE1 genes in B. rapa, B. nigra and B. juncea(group1); and between two FAE1 genes in B. nupus and B. oleracea ( group2). However, FAE1 genes homology was lower between group1 and group2. 3.4 Integrated analysis on the correlation of SNP sites, the base difference at position 968(C/T) in BJFAE1.1 was related to EACs in B. juncea . The base was C in the high EAC varieties and T in the low EAC varieties. The base mutation at position 968 (C→T) in BJFAE1.1 led to the amino acid mutation from threonine(Thr) to isoleucine(Ile)at position 323 in amino acid sequence, which may be the main reason leading to E1→e1. No SNP was found in the BjFAE1.2 which brought in the mutation E2→e2.3.5 BJFAE1 sequence analysis indicated that the genotype of medium EAC line of the yellow mustard in Northern Shaanxi was e1e1E2E2, suggesting the mutation on locus E1 led to the medium EAC of yellow mustard in Northern Shaanxi. For BJFAE1 gene there was only one nucleotide difference on site 1265 between low EAC mutant (1278-3) and low EAC cultivar (4923) from abroad. On site 1265 the nucleotide base of former BJFAE1.1 gene was C, while the latter had two types of nucleotide base(T or C).