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In this study, four varieties of milk vetch were resequenced, and 15 pairs of SSR primers with good polymorphism were developed based on the sequencing results.
A total of 52 alleles were amplified by these 15 pairs of SSR primers, and the average number of effective alleles amplified by each pair of primers was 2.69, and the average polymorphism information content was 0.50.
These 15 pairs of SSR primers were used to analyze the genetic diversity of 103 Chinese milk vetch germplasm resources and draw cluster maps.
When the genetic similarity coefficient was 0.88, all milk vetch germplasm resources could be divided into three categories, and the fitting co-correlation coefficient was 0.64, indicating that the results were true and reliable.
Chinese milk vetch ISSR analysis and molecular marker techniques
Molecular markers are an important means to study plant genetic diversity, and the most commonly used molecular marker methods include restriction amplification fragment length polymorphisms, random amplification polymorphisms, amplification fragment length polymorphisms, correlation sequence amplification polymorphisms, simple repeats, single nucleotide polymorphisms, etc.
Simple repeats, also known as SSR markers, have been widely used to analyze the genetic diversity of various crops, horticultural plants, and grass plants.
The principle is to design specific primers according to the highly conserved sequences at both ends of the microsatellite sequence, and amplify the microsatellite fragments through PCR reaction.
Due to the different number of core sequence tandem repeats, the length of the PCR product is also different, and each amplification site represents a pair of alleles at this locus, which can reveal high polymorphisms due to the large variation in the number of SSR repeats.
The fingerprint technology based on molecular marker technology has the characteristics of fast identification speed and reliable results, and has been widely used in variety identification and kinship research.
As the most important green manure crop in the winter fallow field of rice in southern China, a large number of stems, leaves and root stubble decompose after being pressed and returned to the field.
It can also increase the content of soil humus and total nitrogen, activate and accumulate mineral nutrients, and play the role of fertilization and soil modification. In addition, milk vetch has a variety of uses such as ornamental and vegetable, and is an important nectar source and fodder crop.
China is the origin of milk vetch, and is the country with the earliest and largest cultivation area in the world.
Before the founding of the People's Republic of China, milk vetch was planted in the middle and lower reaches of the Yangtze River and the Qinling-Huaihe River, and the wild resources were mainly distributed at about 33 degrees north latitude.
After the founding of the People's Republic of China, the country promoted green manure planting, and the planting area of milk vetch developed on a large scale, extending to the south of Henan, the north of Jiangsu, the south to the Liangguang region, the west to the Sichuan-Chongqing region, and the east to Zhejiang, Fujian and other eastern coastal areas.
In the 40s of the 20th century, milk vetch was widely cultivated as a green manure crop in northern Japan, and its southern neighbors Myanmar and Vietnam also had a history of introducing milk vetch.
There are few studies on the genetic diversity of milk vetch germplasm resources, and some studies have analyzed the polymorphisms of 9 milk vetch varieties to clarify the appropriate sampling amount.
In addition, 6 pairs of SSR primers were screened to amplify 18 loci with stable and obvious bands, and these 9 milk vetch varieties were divided into two groups.
Experts optimized the ISSR-PCR reaction system of milk vetch, and selected 15 pairs of ISSR primers from 100 ISSR primers published by Columbia University.
The optimal annealing temperature was clarified, and the reaction system suitable for the ISSR analysis of milk vetch was determined, and the genetic diversity of 11 milk vetch resources was analyzed.
A total of 131 RAPD bands were amplified by 11 pairs of 10-mer primers, and the clustering results were clearly divided into three categories.
In general, there is no reliable SSR technique that can comprehensively and systematically analyze the genetic diversity of milk vetch germplasm resources.
In this study, high-throughput sequencing technology was used to resequence the genome of milk vetch, and polymorphic SSR molecular markers of milk vetch were developed, and the genetic diversity of 103 milk vetch germplasm resources was analyzed at the molecular level, and a molecular fingerprint was constructed to distinguish the germplasm resources of milk vetch.
This study can provide a basis for the identification and utilization of milk vetch germplasm resources and subsequent breeding work, especially the selection of parents in hybrid breeding.
Development of milk vetch SSR molecular markers
DNA was extracted from the local varieties of milk vetch "Yuezi No. 1", "Ningbo Daqiao", "Yujiang Daye" and the national variety "Shengzhong", and Guangzhou Gidiao Biotechnology Co., Ltd. was entrusted to perform Illumina10X high-throughput sequencing to obtain the original resequencing data.
A total of 36,433 SSR markers of 6 base repeat types were detected in the raw sequencing data, and 33,975 sequences containing SSR markers were detected, of which 2,249 sequences contained two or more SSR markers.
In terms of SSR molecular marker types, there were 15948 and 13393 repeat types, which accounted for 43.8% and 36.8% of the total SSR markers, respectively.
Among them, there were 3868, 1333 and 1177 SSR molecular markers with four-base, five-base and six-base labels, accounting for 10.6%, 3.7% and 3.2% of the total number of SSR labels, respectively, while the single-base repeat type was the least, with only 714 types, accounting for 2.0% of the total number of SSR labels.
Among all SSR molecular markers, the two-base repeat motif AT/AT and the three-base repeat motif AAG/CTT had the most occurrences, with 7040 and 7039 SSR markers, followed by the two-base repeat motif AG/CT, with 6698 SSR markers.
In addition, A/T with 570 SSR markers was the most abundant in single-base repeat motifs, and AC/GT was more abundant in two-base repeat motifs with 2170 SSR markers.
AAT/ATT, ATC/ATG, AAC/GTT, ACC/GGT, AGG/CCT, ACT/AGT were 2138, 1309, 1228, 679, 413 and 295 respectively.
而四碱基重复基元中出现较多的类型是AAAT/ATTT、AAAG/CTTT、AACT/AGTT和AATT/AATT四种，SSR标记数为1442个、485个、358个和326个。
The deletion and insertion sites in the resequencing data were analyzed, and a total of 59 possible differential sites and 117 pairs of primers were obtained.
Six milk vetch materials "Jitian Seed", "Ningbo Daqiao", "Xiangzi 1", "Shengzhong", "Yujiang Daye" and "Xinzi 1" were amplified, and 15 pairs of polymorphic primers with clear bands were selected from 117 pairs of primers.
The polyacrylamide gel map of the amplified bands was read from top to bottom, and the bands with bands at the same transverse position were counted as 1, and the weak bands with no bands or difficult to distinguish were marked as 0, and the original "0,1" matrix of the amplified products was constructed.
A total of 52 polymorphic sites were amplified by 15 pairs of SSR primers for 103 materials, with an average of 3.4 polymorphic sites per primer.
Among them, 6 polymorphic bands were amplified by primer 13, which was the most among the 15 pairs of primers, 2 polymorphic bands were amplified by primers 5, 7, 9 and 14, which was the least among 15 pairs of primers, and the number of amplified bands was between 3~5 in the rest of primers.
A total of 55 alleles were amplified by 15 pairs of primers in 103 milk vetch germplasm resource materials, and the number of effective alleles varied between 1.12~5.66, with an average value of 2.69, and the diversity index varied between 0.22~1.76, with an average value of 1.00.
The content of polymorphism information varied between 0.799~0.101, and the mean value was 0.504, and the diversity index and polymorphism information content were high, indicating that the primer amplification results were polymorphic.
In addition, it can be found that the average expected heterozygosity, diversity index and polymorphism information content of loci with more effective alleles are also relatively high, and the four have a relatively consistent change trend, indicating that the use of these 15 pairs of primers can truly and accurately reflect the genetic diversity of the tested milk vetch germplasm materials.
Cluster analysis of milk vetch germplasm resources
Then, the genetic diversity of 103 milk vetch germplasm resources from the Institute of Soil and Fertilizer Research Institute of Fujian Academy of Agricultural Sciences was analyzed by using the averaging method and the genetic similarity coefficient as the basis, and the kinship dendrogram was drawn.
The genetic similarity coefficient of milk vetch germplasm resources ranged from 0.753 to 1, and the mean value was 0.892, indicating that the genetic relationship between the milk vetch germplasm resources was not far from each other, and the genetic background was relatively simple.
When the genetic similarity coefficient was 0.87, the milk vetch germplasm resources could be divided into three categories, with 80 materials in class A, 16 materials in class B, and 7 materials in class C, and the fitting co-correlation coefficient r=0.641.
The A category of the dendritic cluster map mainly includes local resources and bred varieties produced in Anhui, Hubei, Jiangsu, Guangxi, Sichuan, Shaanxi and Henan, as well as some local resources and bred varieties produced in Jiangxi, Zhejiang and Hunan.
Category B mainly includes 10 local resources and bred varieties from Zhejiang, including 41, 42 and 44, and 2 bred varieties from Hunan.
Category C includes 3 local resources in Jiangxi, 2 local resources in Zhejiang, 1 local resource in Sichuan and 1 local resource in Fujian.
SSR molecular markers are co-dominant markers with high confidence and abundant polymorphisms, and the identification method is simple and cost-effective. In recent years, SSR markers have been widely used in variety identification, genetic relationship analysis, and DNA fingerprint construction.
In this study, based on the genome resequencing results of four Chinese milk vetch landraces with long distances between origins, Prime3 software was used to identify SSR molecular marker sites.