Analysis Of Male Parents And Comparison Of Cross Combinations And Identification Of Superior Individual Plants In The Offspring Of ‘Emei Wenchun’

‘Emei Wenchun’（Camellia sinensis,EW）is one of the earliest germinating tea varieties,which has many advantages such as short dormancy stage,very early germination and high spring tea yield,and it has been widely cultivated in Sichuan.In this study,we focused on 388 progeny of‘Emei Wenchun’natural pollination by reduced genome sequencing using specific locus amplified fragment sequencing（SLAF-seq）to obtain large-scale SNP（single nucleotide polymorphism）molecular markers for genotyping and paternal identification,Comparison of genetic differences in traits such as sprouting index,fruit number,and root-collar diameter among different parent combinations,and the Morphological characteristics and biochemical component analysis were conducted on 10 excellent individual plants with clear parents,which may provide a foundation for new tea strains.The main results are as follows:（1）Genotyping using simplified genomic sequencing technology SLAF-seq,and the sequencing results generated a total of 579.69 Gb raw data.Through comparison with the reference genome of‘Shuchazao’,a total of 2,629,116 SLAF tags were developed in all samples,of which 1,454,567（55.3%）were polymorphic SLAF tags.The average SLAF sequence depth of EW,candidate fathers,and all the offspring was58.03,30.74 and 11.57.A total of 12,336,977 SNP were detected based on SLAF tag alignment to the reference genome.（2）229218 SNPs with data integrity>80%were selected for paternity identification in all materials.Use ASR（Abnormal SNP Ratios）,IBS（Identity by State）,and maximum likelihood method for paternity identification.The variation range of ASR among 388 offspring,maternal parents,and 14 candidate paternal parents is 0.0042～0.0626;The variation range of IBS with the maternal parent（EW）is 0.7281 to 0.9402.This study selected ASR≥0.02 as the exclusion criterion for male parents based on the second smallest ASR range of all offspring and candidate male parents;Based on the IBS range of offspring and maternal EW,it is determined that IBS≤0.72 is the exclusion criterion for paternal parents.For 14 candidate male parents,11 to 229 offspring each met the ASR standard,and 0 to 220offspring met the IBS standard.On this basis,572 high-quality SNPs were further screened based on allele frequency,sequencing depth and linkage relationship,and parent analysis was carried out using Cervus 3.0 software.The critical Trio（"offspring Emei spring male parent",triplet）LOD value with 99%confidence in parental distribution was estimated to be 0.Results At 99%confidence,a total of 297 offspring matched the most likely parent.The common result of the three methods is taken as the final paternity test result.The paternity of 294（75.8%）offspring was determined,of which 217（55.9%）offspring possessed the same paternal parent‘Chuanmu 217’,30（7.7%）offspring from‘Chuanhuang 1’,11（2.8%）offspring from EW selfing,8（2.1%）offspring from‘Baiye1’,12（3.1%）offspring from‘Dangui’.The remaining 16（4.1%）progeny were from tea cultivars such as‘Huangjinya’,‘Tieguanyin’.（3）Observations were conducted on the sprouting index,root-collar diameter,and fruit number of natural pollinated offspring of 4-5 year old EW.The results showed that the coefficient of variation of the two-year sprouting value of the EW offspring population was 17.09%and 19.42%,respectively,which was 26.69%smaller than the coefficient of variation of the main stem diameter;The coefficient of variation of real numbers is the highest,reaching 116.20%.Pearson correlation analysis was conducted on traits such as sprouting value,trunk diameter,and knot number.The results showed that there was a highly significant positive correlation（P<0.001）between the two years of sprouting value data and trunk diameter,trunk diameter,and knot number in 2022,with correlation coefficients of 0.588,0.316,and 0.301,respectively.（4）Based on the results of paternal identification,compare five hybrid combinations（EW×CM、EW×BY、EW×DG、EW×CH、EW selfing）in sprouting index,root-collar diameter,and fruit number among the difference,in order to provide reference for the selection of hybrid parents.The results show that the highest average sprouting index in 2021 and 2022 were EW,respectively×DG and EW×CM combination,with EW having the lowest germination value in two years×The CH combination was significantly lower than the first two combinations（P<0.01）;The offspring with the highest average root-collar diameter and sprouting index are EW×CM and EW×BY combination,while the smallest ones are EW selfing.The EW×CH and EW selfing has the greatest variation in the two year sprouting index.The largest variation in root-collar diameter and fruit number was EW selfing.The above results indicate that the germination period of offspring of EW hybrid with early maturing varieties is significantly earlier than that of hybrid with late maturing varieties,and there is a decline in EW selfing.The phenotypic traits of sprout and development value in F₁ generation are close to the mid parent value,and the two-year average relative heritability of sprout and development value of female parent is slightly weaker than that of male parent.This indicates that the father has a significant impact on the F₁generation’s sprouting value.（5）According to the observation results of population phenotype,10 excellent individual plants were selected from the EW×CM for morphological and biochemical trait testing.The results showed that there were 3-7 morphological differences between the 10 offspring and the female parent EW,with an average of 5 traits,including leaf edge serration,leaf base shape,style length,style division position,and pistil to stamen height.The results of biochemical composition analysis showed that the content range of water extracts from the spring and summer shoots of the candidate individual plants was 39.12%～42.07%and 38.32%～41.67%,respectively.The tea polyphenol content was 15.35%～18.24%and 19.55%～24.90%,respectively.The caffeine content was3.55%～4.72%and 3.84%～5.06%,and the free amino acid content was 3.06%～5.48%and 1.28%～2.63%,respectively.The total catechin content was 11.63%～15.43%and16.64%～22.29%,respectively,The phenol ammonia ratio is 2.84～5.54 and 7.44～19.46,respectively.The free amino acids in spring are higher than those in summer;The total amount of tea polyphenols,caffeine,and catechins in summer is higher than that in spring.Among the main biochemical components in spring and summer,the coefficient of variation of total free amino acids is the highest（with an average of 17.42%）,indicating a significant variation in the total free amino acid content of the population.Individual plants with higher amino acid content can be selected from this population.Among them,The spring amino acid content（5.48%）and water extract content（42.07%）of single plant 218 are the highest,and the tea polyphenols（15.60%）and catechins content（12.09%）are relatively low;The catechin quality index（1189.52）of individual plant 212 is the highest;In addition,there are 5 individual plants with amino acid content exceeding 4.0%.The above individual plants have the characteristics of good growth potential,early germination period,and high amino acid content,and have breeding potential.In summary,the results of this study provide references or foundations for the design and identification of natural hybrid breeding of tea trees,selection of hybrid parents,selection of new strains,and application for variety rights protection.