基于BSA-seq技术对豌豆花色基因的精细定位

BSA-seq技术在挖掘农艺性状相关的新基因中已被广泛应用,随着豌豆首个参考基因组问世,将BSA-seq技术结合豌豆基因组的基因定位策略势在必行。本研究利用紫花亲本G0004562、白花亲本G0002930以及F2群体,通过BSA-seq技术对豌豆花色基因进行初步定位,获得31.42Mb定位区间,再通过设计InDel分子标记分析进一步缩小定位区间,最终将目标基因定位在包含19个基因的0.99 Mb区间内,通过基因注释信息推测出Psat6g060480.1为豌豆花色候选基因。本研究结果验证了BSA-seq技术快速高效定位豌豆花色基因的可行性,为利用该技术挖掘豌豆其他重要农艺性状相关基因奠定了基础。

西南地区救荒野豌豆种子性状与萌发特性鉴定

救荒野豌豆(Vicia sativa L.)是一种粮菜兼用的食用豆类作物,也是重要的绿肥和优质牧草,具有重要经济价值和显著生态优势。目前关于西南地区救荒野豌豆资源的种子形态鉴定及萌发特性等相关研究存在空白。为了更好的保护和利用救荒野豌豆珍稀濒危野生种质资源,本研究对我国西南地区贵州、四川和云南三省的救荒野豌豆野生种质资源开展了调查收集,共调查了20个县(区),收集到106份形态多样、耐逆性强的野生种质资源。对这些资源的百粒重、粒长和粒宽等8个种子性状进行了鉴定评价,发现百粒重变异系数最大(92%),百粒重最大的来自云南(9.18 g),最小的来自贵州(0.31 g)。同时发现云南地区的救荒野豌豆与贵州和四川地区的资源在8个性状上均有显著差异。此外,对来自3个地区的3份代表性种质资源开展萌发特性研究,发现机械破皮和浓硫酸处理30 min均可有效打破休眠,显著提高救荒野豌豆野生种质资源的种子萌发率。以上研究结果为我国救荒野豌豆珍稀濒危野生资源的保护利用提供了重要理论基础和技术保障。

Construction of SNP genetic maps based on targeted next-generation sequencing and QTL mapping of vital agronomic traits in faba bean(Vicia faba L.)

Owing to the limitation of a large genome size(~13 Gb),the genetic and gene mapping studies on faba bean(Vicia faba L.)are lagging far behind those for other legumes.In this study,we selected three purified faba bean lines(Yundou 8137,H0003712,and H000572)as parents and constructed two F2 populations.These two F2 populations,namely 167 F2 plants in Pop1(Yundou 8137×H0003712)and 204 F2 plants in Pop2(H000572×Yundou 8137),were genotyped using a targeted next-generation sequencing(TNGS)genotyping platform,and two high-density single nucleotide polymorphisms(SNP)genetic linkage maps of faba bean were constructed.The map constructed from Pop1 contained 5103 SNPs with a length of 1333.31 cM and an average marker density of 0.26 cM.The map constructed from Pop2 contained 1904 SNPs with a greater length of 1610.61 cM.In these two F2 populations,QTL mapping identified 98 QTLs for 14 agronomic traits related to the flowers,pods,plant types and grains.The two maps were then merged into an integrated genetic linkage map containing 6895 SNPs,with a length of 3324.48 cM.These results not only lay the foundation for fine mapping and map-based cloning of related genes,but can also accelerate the molecular marker-assisted breeding of faba bean.

低异交率短翼瓣型蚕豆材料的发掘及利用

蚕豆属常异花授粉作物,平均异交率30%左右,高异交率导致其在推广应用中品种一致性、稳定性降低。受媒介及气候因素影响,由传统种质中选育得到的低异交率品种稳定性差。选育具有特殊花器结构的蚕豆品种,减少虫媒觅食授粉,可降低自然异交率,使品种一致性、稳定性得以稳定保持。本研究从云南地方资源中发掘自然变异短翼瓣型蚕豆材料1份,命名为81-37。短翼瓣型花器可避免虫媒与花器接触,即短翼瓣花器结构不利于虫媒驻足采蜜,大幅减少虫媒为介质的异交现象,实现自花授粉,使异交率由30%左右降低至5%以内。以短翼瓣型蚕豆材料、普通型蚕豆材料为亲本进行杂交或回交,从子代中选育具有短翼瓣型花器结构的高产优质蚕豆品种进行推广应用,使品种熟期、花色、种皮种脐颜色、子叶颜色、株高、荚形及营养品质得以稳定保持,提高农产品质量,稳固产量;使品种抗逆性、适用性得以稳定保持,增加原种使用年限,推进产业发展。为蚕豆种质资源保存、品种选育及良种繁育提供优良材料及技术支撑。

重庆地区豌豆(Pisum sativum L.)种质资源收集与多样性分析

"第三次全国农作物种质资源普查与收集行动"重庆项目组于2015—2017年历时3年,从重庆19个区县收集豌豆地方种质资源56份。豌豆种质资源在重庆地区分布呈现出范围广、海拔跨度大的特点。本研究对上述豌豆资源的14个性状进行了遗传多样性分析,结果表明该批资源多样性丰富,其中多样性指数最高的质量性状和数量性状分别是荚型和百粒重;变异系数最大的是分枝数。聚类分析将该批豌豆资源划分为5大类群,第Ⅰ类群为大籽粒和食荚型材料;第Ⅱ类群为高产、中粒型材料;第Ⅲ类群为直立型、早熟型材料;第Ⅳ类群为叶用型兼食荚型材料;第Ⅴ类群为籽粒食用型材料。这些豌豆资源经重庆不同地区和民族农民长年选择后,在抗逆性、食荚性、食叶性、籽粒特性等方面发展出了独特性状,利用好这些资源将为今后的豌豆产业发展注入新的多样性。

Identification and Analysis of Genetic Diversity Structure Within Pisum Genus Based on Microsatellite Markers

To assesse the genetic diversity among wild and cultivated accessions of 8 taxonomic groups in 2 species, and 5 subspecies under Pisum genus, and to analyze population structure and their genetic relationships among various groups of taxonomy, the study tried to verify the fitness of traditionally botanical taxonomic system under Pisum genus and to provide essential information for the exploration and utilization of wild relatives of pea genetic resources. 197 Pisum accessions from 62 counties of 5 continents were employed for SSR analysis using 21 polymorphic primer pairs in this study. Except for cultivated field pea Pisum sativum ssp. sativum var. sativum （94 genotypes）, also included were wild relative genotypes that were classified as belonging to P. fulvum, P. sativum ssp.abyssinicum, P. sativum ssp. asiaticum, P. sativum ssp. transcaucasicum, P. sativum ssp. elatius var. elatius, P. sativum ssp. elatius var. pumilio and P. sativum ssp. sativum var. arvense （103 genotypes）. The PCA analyses and 3-dimension PCA graphs were conducted and drawn by NTSYSpc 2.2d statistical package. Nei78 genetic distances among groups of genetic resources were calculated, and cluster analysis using UPGMA method was carried out by using Popgene V1.32 statistical package, the dendrogram was drawn by MEGA3.1 statistical package. Allelic statistics were carried out by Popgene V1.32. The significance test between groups of genotypes was carried out by Fstat V2.9.3.2 statistical package. 104 polymorphic bands were amplified using 21 SSR primer pairs with unambiguous unique polymorphic bands. 4.95 alleles were detected by each SSR primer pair in average, of which 65.56% were effective alleles for diversity. PSAD270, PSAC58, PSAA18, PSAC75, PSAA175 and PSAB72 were the most effective SSR pairs. SSR alleles were uniformly distributed among botanical taxon units under Pisum genus, but significant difference appeared in most pairwise comparisons for genetic diversity between taxon unit based groups of genetic resources. Genetic diversity level of wild species P. fulvum was much lower than the cultivated species P. sativum. Under species P. sativum, P. sativum ssp. sativum var. sativum and P. sativum ssp. asiaticum were the highest in gentic diversity, followed by P. sativum ssp. elatius var. elatius and P. sativum ssp. transcaucasicum, P. sativum ssp. elatius var. pumilio, P. sativum ssp. sativum vat. arvense and P. sativum ssp. abyssinicum were the lowest. Four gene pool clusters were detected under Pisum genus by using PCA analysis. Gene pool ＂fulvum＂ mainly consisted of wild species Pisum fulvum, gene pool ＂abyssinicum＂ mainly consisted of P. sativum ssp. abyssinicum, and gene pool ＂arvense＂ mainly consisted of P. sativum ssp. sativum var. arvense. While gene pool ＂sativum＂ were composed by 5 botanical taxon units, they are P. sativum ssp. asiaticum, P. sativum ssp. elatius var. elatius, P. sativum ssp. transcaucasicum, P. sativum ssp. elatius var. pumilio and P. sativum ssp. sativum var. sativum. ＂sativum＂ gene pool constructed the primary gene pool of cultivated genetic resources; ＂fulvum＂ gene pool, ＂abyssinicum＂ gene pool and ＂arvense＂ gene pool together constructed the secondary gene pool of cultivated genetic resources. Pairwise Nei78 genetic distance among botanical taxon based groups of pea genetic resources ranged from 7.531 to 35.956, 3 large cluster groups were identified based on the UPGMA dendrogram. Group Ⅰ equals to ＂sativum＂ and ＂arvense＂ gene pools, Group Ⅱ equals to ＂abyssinicum＂ gene pool, and Group Ⅲ equals to ＂fulvum＂ gene pool. The UPGMA clustering results generally supporting the PCA clusting results. There were significant differences among most botanical groups under Pisum genus, with clear separation of four gene pools for genetic diversity structure. The research results partially support the traditional botanical taxonomy under Pisum genus, and pointed out its advantage and shortcoming. In order to broaden the genetic bases of pea varieties, the genetic potentials in the four gene pools should be thoroughly exploited.

通过CRISPR/Cas9建立豌豆基因组大片段敲除体系

豌豆是世界上主要食用豆类作物之一,因其丰富的营养价值和良好的生态价值而受到越来越多的重视。CRISPR/Cas9作为一种生物育种的新型技术手段,已广泛应用于各种作物的品种改良中,但在豌豆中的应用报道非常少见。本研究以“中豌6号”品种为试验材料,采用CRISPR/Cas9技术手段成功地实现了豌豆基因组中Psat01G0240600-T1、Psat03G0303300-T1和Psat03G0304700-T1基因的大片段缺失,其大片段敲除效率分别是24.1%、13.0%和3.6%。进一步对结果分析发现,不同靶点的编辑效率相差较大,并且大片段缺失的效率取决于2个靶点中较低编辑效率的靶点。本研究利用发状根体系探索了CRISPR/Cas9在豌豆中的应用,首次在豌豆中实现了大片段敲除,对于豌豆的研究具有重要意义。